Anti-serum albumin antibodies

ABSTRACT

The invention relates to anti-serum albumin antibodies and multi-specific binding proteins comprising the same. The invention also relates to pharmaceutical compositions comprising the antibodies or multi-specific binding proteins, expression vectors and host cells for making the antibodies or multi-specific binding proteins, and methods of use of the antibodies or multi-specific binding proteins in treatment of diseases or disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 62/946,932, filed on Dec. 11, 2019, thedisclosure of which is hereby incorporated by reference in its entiretyfor all purposes.

FIELD OF THE INVENTION

The invention relates to anti-serum albumin antibodies andmulti-specific binding proteins comprising the same. The invention alsorelates to pharmaceutical compositions comprising these antibodies ormulti-specific binding proteins, expression vectors and host cells formaking these antibodies or multi-specific binding proteins, and methodsof use of these antibodies or multi-specific binding proteins intreatment of diseases or disorders.

BACKGROUND

Serum albumin is the most abundant protein in serum. It has highstability, solubility, and a long circulatory half-life. Polypeptidesthat bind serum albumin, such as antibodies, have been developed toincrease the circulatory half-life of therapeutic proteins. Althoughsignificant developments have been made, there remains a need for newand useful anti-serum albumin antibodies and multi-specific bindingproteins having improved pharmacokinetic properties.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the development of newantibodies that bind serum albumin. Also provided are multi-specificbinding proteins comprising a first domain that binds a first targetprotein expressed on a target cell, such as CD19 (e.g., human CD19),and/or a second domain that binds a second target protein expressed onan immune effector cell, such as CD3 (e.g., human CD3), and a thirddomain that binds serum albumin (e.g., human serum albumin), wherein thethird domain are derived from these new antibodies. These domains arelinked in certain manners for favorable therapeutic efficacy and in vivohalf-life. The multi-specific binding proteins are useful for treatingdiseases and disorders associated with aberrant cells expressing thefirst target protein, such as certain B-cell hematologic malignancies.

Accordingly, in one aspect, the present invention provides anantigen-binding site that binds human serum albumin, comprising a VHcomprising complementarity determining regions HCDR1, HCDR2, and HCDR3,wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences ofSEQ ID NOs: 184, 409, and 411, respectively, but not SEQ ID NOs: 129,133, and 135, respectively.

In certain embodiments, the HCDR1, HCDR2, and HCDR3 comprise the aminoacid sequences of SEQ ID NOs: 184, 185, and 187, respectively, but notSEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, theHCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs:189, 190, and 192, respectively, but not SEQ ID NOs: 129, 133, and 135,respectively. In certain embodiments, the HCDR1, HCDR2, and HCDR3comprise the amino acid sequences of SEQ ID NOs: 189, 193, and 195,respectively, but not SEQ ID NOs: 129, 133, and 135, respectively. Incertain embodiments, the HCDR1, HCDR2, and HCDR3 comprise the amino acidsequences of SEQ ID NOs: 123, 124, and 126, respectively. In certainembodiments, the VH comprises an amino acid sequence at least 85%, atleast 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:121. In certain embodiments, the antigen-binding site binds human serumalbumin with a K_(D) lower than or equal to 10 nM. In certainembodiments, the antigen-binding site binds protein A with a K_(D) lowerthan or equal to 2 nM. The antigen-binding site of any one of claims1-9, wherein the antigen-binding site has a melting temperature greaterthan or equal to 60° C.

In another aspect, the instant disclosure provides a multi-specificbinding protein comprising: (a) a first antigen-binding site that bindsa first target protein expressed on a target cell (e.g., human CD19);(b) a second antigen-binding site that binds a second target proteinexpressed on an immune effector cell (e.g., human CD3); and (c) a thirdantigen-binding site that binds human serum albumin, wherein the thirdantigen-binding site is an antigen-binding site that binds human serumalbumin disclosed herein.

In certain embodiments, the multi-specific binding protein comprises asingle polypeptide chain. In certain embodiments, the thirdantigen-binding site is not positioned between the first antigen-bindingsite and the second antigen-binding site in the polypeptide chain.

In certain embodiments, the third antigen-binding site is positionedN-terminal to both the first antigen-binding site and the secondantigen-binding site in the polypeptide chain. In certain embodiments,the third antigen-binding site is positioned N-terminal to the firstantigen-binding site, and the first antigen-binding site is positionedN-terminal to the second antigen-binding site in the polypeptide chain.In certain embodiments, the third antigen-binding site is positionedN-terminal to the second antigen-binding site, and the secondantigen-binding site is positioned N-terminal to the firstantigen-binding site in the polypeptide chain.

In certain embodiments, the third antigen-binding site is positionedC-terminal to both the first antigen-binding site and the secondantigen-binding site in the polypeptide chain. In certain embodiments,the first antigen-binding site is positioned N-terminal to the secondantigen-binding site, and the second antigen-binding site is positionedN-terminal to the third antigen-binding site in the polypeptide chain.In certain embodiments, the second antigen-binding site is positionedN-terminal to the first antigen-binding site, and the firstantigen-binding site is positioned N-terminal of the thirdantigen-binding site in the polypeptide chain.

In certain embodiments, the first antigen-binding site is positionedN-terminal to the third antigen-binding site, and the thirdantigen-binding site is positioned N-terminal to the secondantigen-binding site in the polypeptide chain. In other embodiments, thesecond antigen-binding site is positioned N-terminal to the thirdantigen-binding site, and the third antigen-binding site is positionedN-terminal binding protein the first antigen-binding site in thepolypeptide chain.

In certain embodiments, the first antigen-binding site comprises asingle-chain variable fragment (scFv). In certain embodiments, the thirdantigen-binding site comprises a single domain antibody (sdAb). Incertain embodiments, the second antigen-binding site comprises an scFv.

In certain embodiments, the second antigen-binding site binds humanCD3ε. In certain embodiments, the second antigen-binding site bindshuman CD3ε with a K_(D) in the range of 1-100 nM.

In certain embodiments, the second antigen-binding site comprises a VHcomprising complementarity determining regions HCDR1, HCDR2, and HCDR3,and a VL comprising complementarity determining regions LCDR1, LCDR2,and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3comprise the amino acid sequences set forth in SEQ ID NOs: 415, 416,418, 419, 420, and 421, respectively. In certain embodiments, the VHcomprises an amino acid sequence at least 85%, at least 90%, at least95%, at least 99%, or 100% identical to SEQ ID NO: 412, and the VLcomprises an amino acid sequence at least 85%, at least 90%, at least95%, at least 99%, or 100% identical to SEQ ID NO: 413. In certainembodiments, the antigen-binding site comprises the amino acid sequenceof SEQ ID NO: 422 or 423.

In certain embodiments, the second antigen-binding site comprises a VHcomprising complementarity determining regions HCDR1, HCDR2, and HCDR3,and a VL comprising complementarity determining regions LCDR1, LCDR2,and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3comprise the amino acid sequences set forth in SEQ ID NOs: 415, 416,426, 419, 420, and 421, respectively. In certain embodiments, the VHcomprises an amino acid sequence at least 85%, at least 90%, at least95%, at least 99%, or 100% identical to SEQ ID NO: 424, and the VLcomprises an amino acid sequence at least 85%, at least 90%, at least95%, at least 99%, or 100% identical to SEQ ID NO: 413. In certainembodiments, the antigen-binding site comprises the amino acid sequenceof SEQ ID NO: 427 or 428.

In certain embodiments, the second antigen-binding site comprises a VHcomprising complementarity determining regions HCDR1, HCDR2, and HCDR3,and a VL comprising complementarity determining regions LCDR1, LCDR2,and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3comprise the amino acid sequences set forth in SEQ ID NOs: 415, 431,418, 419, 420, and 432, respectively. In certain embodiments, the VHcomprises an amino acid sequence at least 85%, at least 90%, at least95%, at least 99%, or 100% identical to SEQ ID NO: 429, and the VLcomprises an amino acid sequence at least 85%, at least 90%, at least95%, at least 99%, or 100% identical to SEQ ID NO: 430. In certainembodiments, the antigen-binding site comprises the amino acid sequenceof SEQ ID NO: 433 or 434.

In certain embodiments, at least two adjacent antigen-binding sites areconnected by a peptide linker. In certain embodiments, each of theadjacent antigen-binding sites are connected by a peptide linker. Incertain embodiments, the peptide linker comprises the amino acidsequence of SEQ ID NO: 298, 299, or 302. In certain embodiments, thepeptide linker consists of the amino acid sequence of SEQ ID NO: 298,299, or 302.

In certain embodiments, the multi-specific binding protein does notcomprise an antibody Fc region. In certain embodiments, the molecularweight of the multi-specific binding protein is at least 65 kD. Incertain embodiments, the serum half-life of the multi-specific bindingprotein is at least 24, 36, 48, or 60 hours.

The instant disclosure also provides an antibody comprising anantigen-binding site that binds human serum albumin disclosed herein.

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising: (a) a multi-specific binding protein or anantibody disclosed herein; and (b) a pharmaceutically acceptablecarrier.

The instant disclosure also provides an isolated polynucleotide encodinga multi-specific binding protein or an antibody disclosed herein. Inaddition, the instant disclosure provides a vector comprising thepolynucleotide disclosed herein, and a recombinant host cell comprisingthe polynucleotide or vector disclosed herein.

The instant disclosure also provides a method of producing amulti-specific binding protein or an antibody, the method comprisingculturing a host cell disclosed herein under suitable conditions thatallow expression of the multi-specific binding protein or the antibody.In certain embodiments, the method further comprises isolating themulti-specific binding protein or the antibody. In certain embodiments,the method further comprises formulating the isolated multi-specificbinding protein or antibody with a pharmaceutically acceptable carrier.

In addition, the instant disclosure provides a method of stimulating animmune response against a target cell, the method comprising exposingthe cell and a T lymphocyte to a multi-specific binding protein, anantibody, or a pharmaceutical composition disclosed herein.

The instant disclosure also provides a method of treating a hematologiccancer in a subject in need thereof, the method comprising administeringto the subject an effective amount of a multi-specific binding protein,an antibody, or a pharmaceutical composition disclosed herein. Incertain embodiments, the hematologic cancer is a B-cell hematologicmalignancy.

In addition, the instant disclosure provides a complex comprising a Tcell expressing CD3, a B cell expressing CD19, and a multi-specificbinding protein disclosed herein, wherein the multi-specific bindingprotein simultaneously bind both the T cell and the B cell. In certainembodiments, the complex further comprises serum albumin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of six domain arrangements ofsingle-chain multi-specific binding proteins. The CD19 binding domain inthe form of an scFv, the CD3 binding domain in the form of an scFv, andthe HSA binding domain in the form of an sdAb are linked in differentorientations. The top of each construct represents the N-terminus andthe bottom of each construct represents the C-terminus of a givenpolypeptide chain.

DETAILED DESCRIPTION

The present invention is based, in part, upon the development of newantibodies that bind serum albumin. Also provided are multi-specificbinding proteins comprising a first domain that binds a first targetprotein expressed on a target cell, such as CD19 (e.g., human CD19),and/or a second domain that binds a second target protein expressed onan immune effector cell, such as CD3 (e.g., human CD3), and a thirddomain that binds serum albumin (e.g., human serum albumin), wherein thethird domain is derived from these new antibodies. These domains arelinked in certain manners for favorable therapeutic efficacy and in vivohalf-life. The multi-specific binding proteins are useful for treatingdiseases and disorders associated with aberrant cells expressing thefirst target protein, such as certain B-cell hematologic malignancies.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term “multi-specific binding protein” refers to a protein or proteinconjugate capable of binding two or more different targets (e.g., two ormore different antigens or two or more different epitopes of the sameantigen). For example, the multi-specific binding protein can bind twoor more different targets through two or more different binding domains.The structure and/or function of the multi-specific binding protein canbe based on the structure and/or function of an antibody, e.g., afull-length or whole immunoglobulin molecule, an antibody heavy chainvariable domain (VH) and/or light chain variable domain (VL), and/or asingle chain antibody. In one example, each one of the binding domainsof a multi-specific binding protein according to the invention comprisesthe minimum structural requirements of an antibody which allow for thetarget binding. This minimum requirement may be, e.g., defined by thepresence of at least the three heavy chain CDRs (i.e. CDR1, CDR2 andCDR3 of the VH domain) and/or the three light chain CDRs (i.e. CDR1,CDR2 and CDR3 of the VL domain). An alternative approach to defining theminimal structural requirements of an antibody is defining the epitopeof a specific target to which the antibody binds, or by referring to aknown antibody with which the antibody competes to bind to the sameepitope that the known antibody binds. The antibodies on which theconstructs according to the invention are based include for examplemonoclonal, recombinant, chimeric, deimmunized, humanized and humanantibodies.

Any one of the binding domains of a multi-specific binding proteinaccording to the invention may comprise the above referred groups ofCDRs. Those CDRs may be comprised in the framework of a VH and/or VL. Fdfragments, for example, have two VH domains and often retain someantigen-binding function of the intact antigen-binding domain.Additional examples for formats of antibody fragments, antibody variantsor binding domains include: (1) a Fab fragment, a monovalent fragmenthaving the VL, VH, CL and CH1 domains; (2) a F(ab′)₂ fragment, abivalent fragment having two Fab fragments linked by a disulfide bridgeat the hinge region; (3) an Fd fragment having the two VH and CH1domains; (4) an Fv fragment having the VL and VH domains of a single armof an antibody; (5) a dAb fragment (Ward et al., (1989) Nature341:544-546), which has a VH domain; (6) an isolated complementaritydetermining region (CDR); and (7) a single chain Fv (scFv), which may bederived, for example, from an scFv-library. Exemplary formats ofmulti-specific binding proteins according to the invention are describedin, e.g., WO2000006605A2, WO2005040220A1, WO2008119567A2,WO2010037838A2, WO2013026837A1, WO 2013026833A1, US 20140308285A1,US20140302037A1, WO2014144722A2, WO2014151910A1, and WO2015048272A1.

Multi-specific binding proteins according to the invention may alsocomprise modified fragments of antibodies, also called antibodyvariants, such as di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab,Fab₂, Fab₃, diabodies, single chain diabodies, tandem diabodies(Tandab's), tandem di-scFv, tandem tri-scFv, “multibodies” such astriabodies or tetrabodies, or single-domain antibodies such asnanobodies or single variable domain antibodies comprising a singlevariable domain, which might be VH (also called VHH in the context of ansdAb) or VL, that specifically bind an antigen or epitope independentlyof other V regions or domains.

As used herein, the terms “single-chain Fv,” “single-chain antibody,”and “scFv” refer to a single-polypeptide-chain antibody fragment thatcomprise the variable regions from both the heavy and light chains, butlack the constant regions. Generally, a single-chain antibody furthercomprises a peptide linker connecting the VH and VL domains whichenables it to form the desired structure to bind to antigen. Singlechain antibodies are discussed in detail by Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods ofgenerating single chain antibodies are known, including those describedin U.S. Pat. Nos. 4,694,778 and 5,260,203; International PatentApplication Publication No. WO 88/01649; Bird (1988) Science242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988)Science 242:1038-1041. In specific embodiments, single-chain antibodiescan also be bispecific, multispecific, human, humanized and/orsynthetic.

Furthermore, the “multi-specific binding protein” described herein canbe a monovalent, bivalent or polyvalent/multivalent construct. Moreover,the “multi-specific binding protein” described herein can include amolecule consisting of only one polypeptide chain, or a moleculesconsisting of more than one polypeptide chain, wherein the chains can beeither identical (homodimers, homotrimers or homo oligomers) ordifferent (heterodimer, heterotrimer or heterooligomer). Examples forthe above identified antibodies and the variants or derivatives thereofare described, for example, in Harlow and Lane, Antibodies a laboratorymanual, CSHL Press (1988); Using Antibodies: a laboratory manual, CSHLPress (1999); Kontermann and Dibel, Antibody Engineering, Springer, 2nded. 2010; and Little, Recombinant Antibodies for Immunotherapy,Cambridge University Press 2009.

The domains of the multi-specific binding protein of the presentinvention may be connected through one or more peptide bonds and/orpeptide linkers. The term “peptide linker” comprises in accordance withthe present invention an amino acid sequence linking two domains. Thepeptide linkers can also be used to fuse the third domain to the otherdomains of the multi-specific binding protein of the invention. Anessential technical feature of such peptide linker is that it does notcomprise any polymerization activity. Among the suitable peptide linkersare those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 orWO198809344A1.

The multi-specific binding proteins of the present invention may be invitro generated multi-specific binding proteins. The term “in vitrogenerated multi-specific binding protein” refers to a multi-specificbinding protein according to the above definition where all or part ofthe variable region (e.g., at least one CDR) is generated by non-immunecell selection, e.g., an in vitro phage display, protein chip or anyother method in which candidate sequences can be tested for theirability to bind to an antigen. The multi-specific binding proteins ofthe present invention may also be generated by genomic rearrangement inan immune cell in an animal. A “recombinant antibody” is an antibodymade through the use of recombinant DNA technology or geneticengineering.

The multi-specific binding protein of the invention may be monoclonal.The term “monoclonal,” as used herein, means that the proteins obtainedfrom a population are substantially homogeneous, i.e., the individualproteins in the population are identical except for naturally occurringmutations and/or post-translation modifications (e.g., isomerizations,amidations) that may be present. In the context of antibodies,monoclonal antibodies are highly specific, being directed against asingle antigenic side or determinant on the antigen, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (orepitopes). The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method.

The multi-specific binding protein of the invention or one or moreantigen-binding site thereof may be affinity matured. In immunology,affinity maturation is the process by which B cells produce antibodieswith increased affinity for antigen during the course of an immuneresponse. With repeated exposures to the same antigen, a host willproduce antibodies of successively greater affinities. Like the naturalprototype, the in vitro affinity maturation is based on the principlesof mutation and selection. Two or three rounds of mutation and selectionusing display methods such as phage display can result in antibodyfragments with affinities in the low nanomolar range.

An amino acid substitution variation can be introduced into themulti-specific binding proteins by substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sides (e.g.,6-7 sides) are mutated to generate all possible amino acid substitutionsat each side. The antibody variants thus generated are displayed in amonovalent fashion from filamentous phage particles as fusions to thegene III product of M13 packaged within each particle. Thephage-displayed variants are then screened for their biological activity(e.g., binding affinity) as herein disclosed. In order to identifycandidate hypervariable region sides for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the bindingdomains. Such contact residues and neighboring residues are candidatesfor substitution according to the techniques elaborated herein. Oncesuch variants are generated, the panel of variants is subjected toscreening as described herein and antibodies with superior properties inone or more relevant assays may be selected for further development.

The multi-specific binding proteins of the present inventionspecifically can comprise “chimeric” antibodies (immunoglobulins) orfragments thereof in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is/are identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. U.S.A., 81:6851-55). Chimeric antibodies of interest herein include “primatized”antibodies comprising variable domain antigen-binding sequences derivedfrom a non-human primate (e.g., Old World Monkey, Ape etc.) or humanconstant region sequences. A variety of approaches for making chimericantibodies have been described. See e.g., Morrison et al. (1985) Proc.Natl. Acad. Sci. U.S.A., 81:6851; Takeda et al. (1985) Nature, 314: 452;U.S. Pat. Nos. 4,816,567; 4,816,397; European Patent No. EP0171496;European Patent Application Publication No. EP0173494; and U.K. PatentNo. GB2177096.

The term “binding domain” or “domain that binds (an antigen)”characterizes in connection with the present invention a domain which(specifically) binds to or interacts with a given target epitope or agiven target side on the target molecules (antigens), e.g. CD19, serumalbumin, and CD3, respectively. The structure and function of the firstbinding domain, the second binding domain, and/or the third bindingdomain can be based on the structure and/or function of an antibody,e.g. of a full-length or whole immunoglobulin molecule. A binding domaincan be drawn from the VH and/or VL or VHH domain of an antibody orfragment thereof. For example, a binding domain can include three lightchain CDRs (i.e., CDR1, CDR2 and CDR3 of the VL domain) and/or threeheavy chain CDRs (i.e., CDR1, CDR2 and CDR3 of the VH domain). A bindingdomain can also include VHH CDRs (i.e., CDR1, CDR2 and CDR3 of the VHHregion).

The terms “variable domain” and “variable region” are usedinterchangeably and refer to the portions of the antibody orimmunoglobulin domains that exhibit variability in their sequence andthat are involved in determining the specificity and binding affinity ofa particular antibody. Variability is not evenly distributed throughoutthe variable domains of antibodies; it is concentrated in sub-domains ofeach of the heavy and light chain variable regions. These sub-domainsare called “hypervariable regions” or “complementarity determiningregions” (CDRs). The more conserved (i.e., non-hypervariable) portionsof the variable domains are called the “framework” regions (FRM or FR)and provide a scaffold for the six CDRs in three dimensional space toform an antigen-binding surface.

In the present invention, any one of the binding domains of themulti-specific binding protein may comprise a single domain antibody(sdAb). A single domain antibody comprises a single, monomeric antibodyvariable domain which is able to bind selectively to a specific antigen,independently of other variable regions or domains. The first singledomain antibodies were engineered from heavy chain antibodies found incamelids, and these are called WEI fragments. Cartilaginous fishes alsohave heavy chain antibodies (IgNAR) from which single domain antibodiescalled V_(NAR) fragments can be obtained. An alternative approach is tosplit the dimeric variable domains from common immunoglobulins e.g.,from humans or rodents into monomers, hence obtaining VH or VL as asingle domain antibody. Although most research into single domainantibodies is currently based on heavy chain variable domains,nanobodies derived from light chains have also been shown to bindspecifically to target epitopes. Examples of single domain antibodiesinclude nanobodies and single variable domain antibodies.

As used herein, the term “antigen-binding site” refers to the part of animmunoglobulin molecule or a derivative or variant thereof thatparticipates in antigen binding. In human antibodies, the antigenbinding site is formed by amino acid residues of the N-terminal variable(“V”) regions of the heavy (“H”) and light (“L”) chains. Three highlydivergent stretches within the V regions of the heavy and light chainsare referred to as “hypervariable regions” which are interposed betweenmore conserved flanking stretches known as “framework regions,” or “FR.”Thus the term “FR” refers to amino acid sequences which are naturallyfound between and adjacent to hypervariable regions in immunoglobulins.In a human antibody molecule, the three hypervariable regions of a lightchain and the three hypervariable regions of a heavy chain are disposedrelative to each other in three dimensional space to form anantigen-binding surface. The antigen-binding surface is complementary tothe three-dimensional surface of a bound antigen, and the threehypervariable regions of each of the heavy and light chains are referredto as “complementarity-determining regions,” or “CDRs.” In certainanimals, such as camels and cartilaginous fish, the antigen-binding siteis formed by a single antibody chain providing a “single domainantibody.” Antigen-binding sites can exist in an intact antibody, in anantigen-binding fragment of an antibody that retains the antigen-bindingsurface, or in a recombinant polypeptide such as an scFv, using apeptide linker to connect the heavy chain variable domain to the lightchain variable domain in a single polypeptide.

As used herein, the term “antibody” refers to a protein or a proteinconjugate that comprises an antigen-binding site. An antibody can bemonospecific or multi-specific (e.g., bispecific).

The terms “a” and “an” as used herein mean “one or more” and include theplural unless the context is inappropriate.

As used herein, the terms “subject” and “patient” refer to an organismto be treated by the methods and compositions described herein. Suchorganisms preferably include, but are not limited to, mammals (e.g.,murines, simians, equines, bovines, porcines, canines, felines, and thelike), and more preferably include humans.

As used herein, the term “effective amount” refers to the amount of acompound (e.g., a compound of the present invention) sufficient toeffect beneficial or desired results. An effective amount can beadministered in one or more administrations, applications or dosages andis not intended to be limited to a particular formulation oradministration route. As used herein, the term “treating” includes anyeffect, e.g., lessening, reducing, modulating, ameliorating oreliminating, that results in the improvement of the condition, disease,disorder, and the like, or ameliorating a symptom thereof.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975).

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present invention that consist essentially of, or consist of, therecited processing steps.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

1. Anti-Serum Albumin Antibodies

In one aspect, the present disclosure provides an antigen-binding sitethat binds serum albumin (e.g., human serum albumin (HSA)) derived fromthe single domain antibodies listed in Table 1. The present disclosurealso provides an antibody comprising the antigen-binding site. The CDRsequences are identified under the Kabat numbering scheme unlessindicated by an asterisk (*).

TABLE 1 Sequences of Exemplary Antibodies That Bind Serum AlbuminAntibody VH and HCDRs CNG-HSA-KVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMTWVRQAPGKGLEWVSSISG 101SGSDTLYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIGGSLSPSSQGTLVTVSS (SEQ ID NO: 121) HCDR1*: FTFSSFGMT (SEQ ID NO: 122)HCDR1: SFGMT (SEQ ID NO: 123) HCDR2: SISGSGSDTLYADSVRG (SEQ ID NO: 124)HCDR3*: TIGGSLSP (SEQ ID NO: 125) HCDR3: GGSLSP (SEQ ID NO: 126)CNG-HSA- KVQLLESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 102SGSDTLYAD S VRGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSPSSQGTLVTVSS (SEQ ID NO: 127) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVRG (SEQ ID NO: 124)HCDR3*: TIGGSLSP (SEQ ID NO: 125) HCDR3: GGSLSP (SEQ ID NO: 126)CNG-HSA- KVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMTWVRQAPGKGLEWVSSISG 103SGSDTLYADSVRGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSPSSQGTLVTVSS (SEQ ID NO: 130) HCDR1*: FTFSSFGMT (SEQ ID NO: 122)HCDR1: SFGMT (SEQ ID NO: 123) HCDR2: SISGSGSDTLYADSVRG (SEQ ID NO: 124)HCDR3*: TIGGSLSP (SEQ ID NO: 125) HCDR3: GGSLSP (SEQ ID NO: 126)CNG-HSA- KVQLLESGGGLVQPGGSLRLSCAASGFTFHSFGMSWVRQAPGKGLEWVSSISG 104SGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 131) HCDR1*: FTFHSFGMS (SEQ ID NO: 132)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 133)HCDR3*: TIGGSLSR (SEQ ID NO: 134) HCDR3: GGSLSR (SEQ ID NO: 135)CNG-HSA- EVQLLESGGGLVQPGGSLRLSCAASGFVFSSFGMSWVRQAPGKGLEWVSSISG 105SGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 136) HCDR1*: FVFSSFGMS (SEQ ID NO: 137)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 133)HCDR3*: TIGGSLSR (SEQ ID NO: 134) HCDR3: GGSLSR (SEQ ID NO: 135)CNG-HSA- KVQLLESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 106SGSDTLYADSVRGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCTIGGSRSISSQGTLVTVSS (SEQ ID NO: 138) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVRG (SEQ ID NO: 124)HCDR3*: TIGGSRSI (SEQ ID NO: 139) HCDR3: GGSRSI (SEQ ID NO: 140)CNG-HSA- KVQLLESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 107SGSDTLYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSIGGSLIRSSQGTLVTVSS (SEQ ID NO: 141) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVRG (SEQ ID NO: 124)HCDR3*: SIGGSLIR (SEQ ID NO: 142) HCDR3: GGSLIR (SEQ ID NO: 143)CNG-HSA- KVQLVESGGGLVQPGGSLRLSCAASGFTFGSFGMSWVRQAPGKGLEWVSSISG 108SGADTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIGGSLRASSQGTLVTVSS (SEQ ID NO: 144) HCDR1*: FTFGSFGMS (SEQ ID NO: 145)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGADTLYADSVKG (SEQ ID NO: 146)HCDR3*: TIGGSLRA (SEQ ID NO: 147) HCDR3: GGSLRA (SEQ ID NO: 148)CNG-HSA- KVQLVESGGGLVQPGNSLRLSCAASGFTFGSFGMSWVRQAPGKGPEWVSSISG 109SGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 149) HCDR1*: FTFGSFGMS (SEQ ID NO: 145)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 133)HCDR3*: TIGGSLSR (SEQ ID NO: 134) HCDR3: GGSLSR (SEQ ID NO: 135)CNG-HSA- KVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 110SGGDTLYADSAKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 150) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGGDTLYADSAKG (SEQ ID NO: 151)HCDR3*: TIGGSLSR (SEQ ID NO: 134) HCDR3: GGSLSR (SEQ ID NO: 135)CNG-HSA- KVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 111SGSDTLYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIGGSLKQSSQGTLVTVSS (SEQ ID NO: 152) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVEG (SEQ ID NO: 153)HCDR3*: TIGGSLKQ (SEQ ID NO: 154) HCDR3: GGSLKQ (SEQ ID NO: 155)CNG-HSA- KVQLLESGGGLVQPGGSLRLSCAASGFTFPSFGMSWVRQAPGKGLEWVSSISG 112SGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSVSKSSRGTLVTVSS (SEQ ID NO: 156) HCDR1*: FTFPSFGMS (SEQ ID NO: 157)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 133)HCDR3*: TIGGSVSK (SEQ ID NO: 158) HCDR3: GGSVSK (SEQ ID NO: 159)CNG-HSA- GVQLLESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 113TGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLRYSSQGTLVTVSS (SEQ ID NO: 160) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGTGSDTLYADSVKG (SEQ ID NO: 161)HCDR3*: TIGGSLRY (SEQ ID NO: 162) HCDR3: GGSLRY (SEQ ID NO: 163)CNG-HSA- KVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 114SGSDTLTADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIGGSLVRSSQGTLVTVSS (SEQ ID NO: 164) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLTADSVKG (SEQ ID NO: 165)HCDR3*: TIGGSLVR (SEQ ID NO: 166) HCDR3: GGSLVR (SEQ ID NO: 167)CNG-HSA- KVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMGWVRQAPGKGLEWVSSISG 115SGSDTLYAPSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTFAGSLSRSSQGTLVTVSS (SEQ ID NO: 168) HCDR1*: FTFSSFGMG (SEQ ID NO: 169)HCDR1: SFGMG (SEQ ID NO: 170) HCDR2: SISGSGSDTLYAPSVKG (SEQ ID NO: 171)HCDR3*: TFAGSLSR (SEQ ID NO: 172) HCDR3: AGSLSR (SEQ ID NO: 173)CNG-HSA- KVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKGPEWVSSISG 116SGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIGGSLRASSQGTLVTVSS (SEQ ID NO: 174) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 133)HCDR3*: TIGGSLRA (SEQ ID NO: 147) HCDR3: GGSLRA (SEQ ID NO: 148)CNG-HSA- KVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 117SGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIGGSLTRSSQGTLVTVSS (SEQ ID NO: 175) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 133)HCDR3*: TIGGSLTR (SEQ ID NO: 176) HCDR3: GGSLTR (SEQ ID NO: 177)CNG-HSA- KVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 118GGSDTLYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIGGSLRASSQGTLVTVSS (SEQ ID NO: 178) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGGGSDTLYADSVRG (SEQ ID NO: 179)HCDR3*: TIGGSLRA (SEQ ID NO: 147) HCDR3: GGSLRA (SEQ ID NO: 148)CNG-HSA- KVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG 119SGSDALYADSAKGRFTISRDNAKTTLYLQMNSLRAEDTAVYYCTIGGSLSPSSQGTLVTVSS (SEQ ID NO: 180) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDALYADSAKG (SEQ ID NO: 181)HCDR3*: TIGGSLSP (SEQ ID NO: 125) HCDR3: GGSLSP (SEQ ID NO: 126)CNG-HSA- KVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKGPEWVSSISG 120SGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLKQSSQGTLVTVSS (SEQ ID NO: 182) HCDR1*: FTFSSFGMS (SEQ ID NO: 128)HCDR1: SFGMS (SEQ ID NO: 129) HCDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 133)HCDR3*: TIGGSLKQ (SEQ ID NO: 154) HCDR3: GGSLKQ (SEQ ID NO: 155)Consensus-1HCDR1*: FX₁FX₂SFGMX₃, wherein X₁ is T or V; X₂ is S, H, G, or P; and X₃ is S, T,(CNG-HSA- or G (SEQ ID NO: 408) 101 to -120)HCDR1: SFGMX, whereinX is S, T, or G (SEQ ID NO: 184)HCDR2: SISGX₁GX₂DX₃LX₄AX₅SX₆X₇G, wherein X₁ is S, T, or G; X₂ is S, A, or G;X₃ is T or A; X₄ is Y or T; X₅ is D or P; X₆ is V or A; and X₇ is K, R, or E(SEQ ID NO: 409)HCDR3*: X₁X₂X₃GSX₄X₅X₆, wherein X₁ is T or S; X₂ is I or F; X₃ is G or A; X₄ is L,R, or V; X₅ is S, I, R, K, V, or T; and X₆ is R, P, I, A, Q, K, or Y(SEQ ID NO: 410)HCDR3: X₁GSX₂X₃X₄, wherein X₁ is G or A; X₂ is L, R, or V; X₃ is S, I, R, K, V,or T; and X₄ is R, P, I, A, Q, K, or Y (SEQ ID NO: 411) Consensus-2HCDR1*: FTFX₁SFGMX₂, wherein X₁ is S, H, G, or P; and X₂ is S, T, or G (SEQ ID(CNG-HSA- NO: 183) 101 to -104,HCDR1: SFGMX, wherein X is S, T, or G (SEQ ID NO: 184) -108 to-112,HCDR2: SISGX₁GX₂DX₃LYAX₄SX₅X₆G, wherein X₁ is S or G; X₂ is S, A, or G; X₃ isand -115 toT or A; X₄ is D or P; X₅ is V or A; and X₆ is K, R, or E (SEQ ID NO: 185)-120)HCDR3*: TX₁X₂GSX₃X₄X₅, wherein X₁ is I or F; X₂ is G or A; X₃ is L or V; X₄ is S,R, K, or T; and X₅ is R, P, A, Q, or K (SEQ ID NO: 186)HCDR3: X₁GSX₂X₃X₄, wherein X₁ is G or A; X₂ is L or V; X₃ is S, R, K, or T; andX₄ is R, P, A, Q, or K (SEQ ID NO: 187) Consensus-3HCDR1*: FTFX₁SFGMX₂, wherein X₁ is S, H, or G; and X₂ is S or T (SEQ ID NO:(CNG-HSA- 188) 101, -102,HCDR1: SFGMX, wherein X is S or T (SEQ ID NO: 189) -104, -109,HCDR2: SISGX₁GSDTLYADSVX₂G, wherein X₁ is S or T; and X₂ is K or R (SEQ ID-113, NO: 190) -116, and-117)HCDR3*: TIGGSLX₁X₂, wherein X₁ is S, R, or T; and X₂ is R, P, Y, or A (SEQ IDNO: 191)HCDR3: GGSLX₁X₂, wherein X₁ is S, R, or T; and X₂ is R, P, Y, or A (SEQ ID NO:192) Consensus-4HCDR1*: FTFX₁SFGMX₂, wherein X₁ is S, H, or G; and X₂ is S or T (SEQ ID NO:(CNG-HSA- 188) 101, -102,HCDR1: SFGMX, wherein X is S or T (SEQ ID NO: 189) -104, -109,HCDR2: SISGSGSDTLYADSVXG, wherein X is K or R (SEQ ID NO: 193)-116, and-117)HCDR3*: TIGGSLXX2, wherein X₁ is S, R, or T; and X₂ is R, P, or A (SEQ ID NO:194)HCDR3: GGSLX₁X₂, wherein X₁ is S, R, or T; and X₂ is R, P, or A (SEQ ID NO: 195)

In certain embodiments, the antigen-binding site that binds serumalbumin of the present invention comprises a VH that comprises an aminoacid sequence at least 60% (e.g., at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%) identical to the VH of an antibody disclosed in Table 1.In certain embodiments, the antigen-binding site comprises the HCDR1,HCDR2, and HCDR3, determined under Kabat (see Kabat et al., (1991)Sequences of Proteins of Immunological Interest, NIH Publication No.91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), JMol Biol 196: 901-917), MacCallum (see MacCallum R M et al., (1996) JMol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7,132-136), or any other CDR determination method known in the art, of theVH sequence of an antibody disclosed in Table 1. In certain embodiments,the antigen-binding site comprises the HCDR1, HCDR2, and HCDR3 sequencesof an antibody disclosed in Table 1. In certain embodiments, theantigen-binding site comprises the VH sequence of an antibody disclosedin Table 1.

Series 1 Constructs

In certain embodiments, the antigen-binding site that binds HSAcomprises a VH comprising HCDR1, HCDR2, and HCDR3 sequences set forth inSEQ ID NOs: 408, 409, and 410, respectively, wherein the antigen-bindingsite does not comprise HCDR1, HCDR2, and HCDR3, sequences set forth inSEQ ID NOs: 128, 133, and 134, respectively. In certain embodiments, theHCDR1 sequence is selected from the group consisting of SEQ ID NOs: 122,128, 132, 137, 145, 157, and 169; the HCDR2 sequence is selected fromthe group consisting of SEQ ID NOs: 124, 133, 146, 151, 153, 161, 165,171, 179, and 181; and/or the HCDR3 sequence is selected from the groupconsisting of SEQ ID NOs: 125, 134, 142, 147, 154, 158, 162, 166, 172,and 176.

In certain embodiments, the antigen-binding site comprises a VHcomprising HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:184, 409, and 411, respectively, wherein the antigen-binding site doesnot comprise HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:129, 133, and 135, respectively. In certain embodiments, the HCDR1sequence is selected from the group consisting of SEQ ID NOs: 123, 129,and 170; the HCDR2 sequence is selected from the group consisting of SEQID NOs: 124, 133, 146, 151, 153, 161, 165, 171, 179, and 181; and/or theHCDR3 sequence is selected from the group consisting of SEQ ID NOs: 126,135, 143, 148, 155, 159, 163, 167, 173, and 177.

In certain embodiments, the antigen-binding site comprises a VHcomprising an amino acid sequence at least 60% (e.g., at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) identical to SEQ ID NO: 121.

In certain embodiments, the antigen-binding site has a higher bindingaffinity to human serum albumin, cynomolgus serum albumin, mouse serumalbumin, and/or protein A relative to an antigen-binding site having VHsequence set forth in SEQ ID NO: 196.

Series 2 Constructs

In certain embodiments, the antigen-binding site that binds HSAcomprises a VH comprising HCDR1, HCDR2, and HCDR3 sequences set forth inSEQ ID NOs: 183, 185, and 186, respectively, wherein the antigen-bindingsite does not comprise HCDR1, HCDR2, and HCDR3, sequences set forth inSEQ ID NOs: 128, 133, and 134, respectively. In certain embodiments, theHCDR1 sequence is selected from the group consisting of SEQ ID NOs: 122,128, 132, 145, 157, and 169; the HCDR2 sequence is selected from thegroup consisting of SEQ ID NOs: 124, 133, 146, 151, 153, 171, 179, and181; and/or the HCDR3 sequence is selected from the group consisting ofSEQ ID NOs: 125, 134, 147, 154, 158, 172, and 176.

In certain embodiments, the antigen-binding site comprises a VHcomprising HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:184, 185, and 187, respectively, wherein the antigen-binding site doesnot comprise HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:129, 133, and 135, respectively. In certain embodiments, the HCDR1sequence is selected from the group consisting of SEQ ID NOs: 123, 129,and 170; the HCDR2 sequence is selected from the group consisting of SEQID NOs: 124, 133, 146, 151, 153, 171, 179, and 181; and/or the HCDR3sequence is selected from the group consisting of SEQ ID NOs: 126, 135,148, 155, 159, 173, and 177.

In certain embodiments, the antigen-binding site comprises a VHcomprising an amino acid sequence at least 60% (e.g., at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) identical to SEQ ID NO: 121.

In certain embodiments, the antigen-binding site has a higher bindingaffinity to human serum albumin relative to an antigen-binding sitehaving VH sequence set forth in SEQ ID NO: 196. In certain embodiments,the antigen-binding site binds human serum albumin with a K_(D) lowerthan or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM, asmeasured by SPR when the antigen-binding site is present as a monomer.In certain embodiments, the antigen-binding site binds human serumalbumin with a K_(D) in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM,1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM, as measured by SPR when theantigen-binding site is present as a monomer.

Series 3 Constructs

In certain embodiments, the antigen-binding site that binds HSAcomprises a VH comprising HCDR1, HCDR2, and HCDR3 sequences set forth inSEQ ID NOs: 188, 190, and 191, respectively, wherein the antigen-bindingsite does not comprise HCDR1, HCDR2, and HCDR3, sequences set forth inSEQ ID NOs: 128, 133, and 134, respectively. In certain embodiments, theHCDR1 sequence is selected from the group consisting of SEQ ID NOs: 122,128, 132, and 145; the HCDR2 sequence is selected from the groupconsisting of SEQ ID NOs: 124, 133, and 161; and/or the HCDR3 sequenceis selected from the group consisting of SEQ ID NOs: 125, 134, 162, 147,and 176.

In certain embodiments, the antigen-binding site comprises a VHcomprising HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:189, 190, and 192, respectively, wherein the antigen-binding site doesnot comprise HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:129, 133, and 135, respectively. In certain embodiments, the HCDR1sequence is selected from the group consisting of SEQ ID NOs: 123 and129; the HCDR2 sequence is selected from the group consisting of SEQ IDNOs: 124, 133, and 161; and/or the HCDR3 sequence is selected from thegroup consisting of SEQ ID NOs: 126, 135, 163, 148, and 177.

In certain embodiments, the antigen-binding site comprises a VHcomprising an amino acid sequence at least 60% (e.g., at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) identical to SEQ ID NO: 121.

In certain embodiments, the antigen-binding site has a higher bindingaffinity to protein A relative to an antigen-binding site having VHsequence set forth in SEQ ID NO: 196. In certain embodiments, theantigen-binding site binds protein A with a K_(D) lower than or equal to2.5 nM or 2 nM, as measured by SPR when the antigen-binding site ispresent as a monomer. In certain embodiments, the antigen-binding sitebinds protein A with a K_(D) in the range of 1-2.5 nM or 1-2 nM, asmeasured by SPR when the antigen-binding site is present as a monomer.

Series 4 Constructs

In certain embodiments, the antigen-binding site that binds HSAcomprises a VH comprising HCDR1, HCDR2, and HCDR3 sequences set forth inSEQ ID NOs: 188, 193, and 194, respectively, wherein the antigen-bindingsite does not comprise HCDR1, HCDR2, and HCDR3, sequences set forth inSEQ ID NOs: 128, 133, and 134, respectively. In certain embodiments, theHCDR1 sequence is selected from the group consisting of SEQ ID NOs: 122,128, 132, and 145; the HCDR2 sequence is selected from the groupconsisting of SEQ ID NOs: 124 and 133; and/or the HCDR3 sequence isselected from the group consisting of SEQ ID NOs: 125, 134, 147, and176.

In certain embodiments, the antigen-binding site comprises a VHcomprising HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:189, 193, and 195, respectively, wherein the antigen-binding site doesnot comprise HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:129, 133, and 135, respectively. In certain embodiments, the HCDR1sequence is selected from the group consisting of SEQ ID NOs: 123 and129; the HCDR2 sequence is selected from the group consisting of SEQ IDNOs: 124 and 133; and/or the HCDR3 sequence is selected from the groupconsisting of SEQ ID NOs: 126, 135, 148, and 177.

In certain embodiments, the antigen-binding site comprises a VHcomprising an amino acid sequence at least 60% (e.g., at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) identical to SEQ ID NO: 121.

In certain embodiments, the antigen-binding site has a higher bindingaffinity to human serum albumin and a higher affinity to protein Arelative to an antigen-binding site having VH sequence set forth in SEQID NO: 196. In certain embodiments, the antigen-binding site binds humanserum albumin with a K_(D) lower than or equal to 10 nM, 9 nM, 8 nM, 7nM, 6 nM, 5 nM, 4 nM, or 3 nM and binds protein A with a K_(D) lowerthan or equal to 2.5 nM or 2 nM, as measured by SPR when theantigen-binding site is present as a monomer. In certain embodiments,the antigen-binding site binds human serum albumin with a K_(D) in therange of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3nM and binds protein A with a K_(D) in the range of 1-2.5 nM or 1-2 nM,as measured by SPR when the antigen-binding site is present as amonomer.

Individual Constructs

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-101. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 122, 124, and 125, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 123, 124, and126, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:121. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 121.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-102. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 124, and 125, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 124, and126, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:127. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 127.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-103. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 122, 124, and 125, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 123, 124, and126, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:130. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 130.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-104. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and135, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:131. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 131.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-105. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 137, 133, and 134, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and135, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:136. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 136.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-106. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 124, and 139, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 124, and140, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:138. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 138.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-107. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 124, and 142, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 124, and143, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:141. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 141.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-108. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 145, 146, and 147, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 146, and148, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:144. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 144.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-109. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 145, 133, and 134, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and135, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:149. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 149.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-110. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 151, and 134, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 151, and135, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:150. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 150.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-111. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 153, and 154, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 153, and155, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:152. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 152.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-112. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 157, 133, and 158, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and159, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:156. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 156.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-113. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 161, and 162, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 161, and163, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:160. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 160.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-114. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 165, and 166, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 165, and167, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:164. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 164.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-115. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 169, 171, and 172, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 170, 171, and173, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:168. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 168.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-116. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 133, and 147, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and148, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:174. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 174.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-117. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 133, and 176, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and177, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:175. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 175.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-118. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 179, and 147, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 179, and148, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:178. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 178.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-119. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 181, and 125, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 181, and126, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:180. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 180.

In certain embodiments, the antigen-binding site that binds serumalbumin is derived from CNG-HSA-120. In certain embodiments, theantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 128, 133, and 154, respectively. Incertain embodiments, the antigen-binding site comprises a VH comprisingHCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and155, respectively. In certain embodiments, the antigen-binding sitecomprises a VH comprising an amino acid sequence at least 60% (e.g., atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:180. In certain embodiments, the VH comprises the amino acid sequence ofSEQ ID NO: 180.

In certain embodiments, the antigen-binding site derived fromCNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108,CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115,CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has ahigher binding affinity to human, cynomolgus, and/or mouse serum albuminrelative to an antigen-binding site having VH sequence set forth in SEQID NO: 196.

In certain embodiments, the antigen-binding site derived fromCNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108,CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115,CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 bindshuman serum albumin with a K_(D) lower than or equal to 10 nM, 9 nM, 8nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM, as measured by SPR when theantigen-binding site is present as a monomer. In certain embodiments,the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102,CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110,CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117,CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 binds human serum albumin witha K_(D) in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM,1-4 nM, or 1-3 nM, as measured by SPR when the antigen-binding site ispresent as a monomer.

In certain embodiments, the antigen-binding site derived fromCNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108,CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115,CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 bindscynomolgus serum albumin with a K_(D) lower than or equal to 9 nM, 8 nM,7 nM, 6 nM, 5 nM, 4 nM, or 3 nM, as measured by SPR when theantigen-binding site is present as a monomer. In certain embodiments,the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102,CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110,CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117,CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 binds cynomolgus serum albuminwith a K_(D) in the range of 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4nM, or 1-3 nM, as measured by SPR when the antigen-binding site ispresent as a monomer.

In certain embodiments, the antigen-binding site derived fromCNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108,CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115,CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 bindsmouse serum albumin with a K_(D) lower than or equal to 100 nM, 90 nM,80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM, as measuredby SPR when the antigen-binding site is present as a monomer. In certainembodiments, the antigen-binding site derived from CNG-HSA-101,CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109,CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116,CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 binds mouse serumalbumin with a K_(D) in the range of 1-100 nM, 1-90 nM, 1-80 nM, 1-70nM, 1-60 nM, 1-50 nM, 1-40 nM, 1-30 nM, 1-20 nM, or 1-10 nM, as measuredby SPR when the antigen-binding site is present as a monomer.

In certain embodiments, the antigen-binding site derived fromCNG-HSA-101, CNG-HSA-103, CNG-HSA-106, CNG-HSA-107, CNG-HSA-108,CNG-HSA-109, CNG-HSA-111, CNG-HSA-113, CNG-HSA-114, CNG-HSA-115,CNG-HSA-116, CNG-HSA-118, or CNG-HSA-120 binds human serum albumin witha first K_(D) and binds mouse serum albumin with a second K_(D), whereinthe ratio of the second K_(D) to the first K_(D) is in the range of0.5-10, 0.5-9, 0.5-8, 0.5-7, 0.5-6, 0.5-5, 0.5-4, 0.5-3, 0.5-2, 0.9-10,0.9-9, 0.9-8, 0.9-7, 0.9-6, 0.9-5, 0.9-4, 0.9-3, 0.9-2, 1-10, 1-9, 1-8,1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. It is understood that anantigen-binding site having a ratio closer to 1 has more similaraffinity to mouse serum albumin relative to affinity to human serumalbumin, which allows assessment of the pharmacokinetics of theantigen-binding site or a protein comprising the same using a mousemodel at higher accuracy.

In certain embodiments, the antigen-binding site derived fromCNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113,CNG-HSA-116, or CNG-HSA-117 has a higher binding affinity to protein Arelative to an antigen-binding site having VH sequence set forth in SEQID NO: 196. It is understood that increased affinity to protein A allowspurification of the antigen-binding site, or a protein that comprisesthe antigen-binding site but not an antibody Fc region, by protein Achromatography. In certain embodiments, the antigen-binding site derivedfrom CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113,CNG-HSA-116, or CNG-HSA-117 binds human serum albumin with a K_(D) lowerthan or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM andbinds protein A with a K_(D) lower than or equal to 2.5 nM or 2 nM, asmeasured by SPR when the antigen-binding site is present as a monomer.In certain embodiments, the antigen-binding site derived fromCNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113,CNG-HSA-116, or CNG-HSA-117 binds human serum albumin with a K_(D) inthe range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or1-3 nM and binds protein A with a K_(D) in the range of 1-2.5 nM or 1-2nM, as measured by SPR when the antigen-binding site is present as amonomer.

In certain embodiments, the antigen-binding site derived fromCNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-116, orCNG-HSA-117 has a higher binding affinity to human serum albumin and ahigher affinity to protein A relative to an antigen-binding site havingVH sequence set forth in SEQ ID NO: 196. In certain embodiments, theantigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104,CNG-HSA-109, CNG-HSA-116, or CNG-HSA-117 binds protein A with a K_(D)lower than or equal to 2.5 nM or 2 nM, as measured by SPR when theantigen-binding site is present as a monomer. In certain embodiments,the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102,CNG-HSA-104, CNG-HSA-109, CNG-HSA-116, or CNG-HSA-117 binds protein Awith a K_(D) in the range of 1-2.5 nM or 1-2 nM, as measured by SPR whenthe antigen-binding site is present as a monomer.

Melting temperature represents the thermostability of theantigen-binding site and can be measured by differential scanningfluorimetry, for example, as described in Durowoju et al. (2017) J. Vis.Exp. (121): 55262. The thermostability of an antibody or fragmentthereof may be enhanced by grafting CDRs onto stable frameworks,introducing non-canonical disulfide bonds, and other mutagenesis, asdescribed in McConnell et al. (2014) MAbs, 6(5): 1274-82; and Goldman etal. (2017) Front. Immunol., 8: 865. In certain embodiments, theantigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103,CNG-HSA-104, CNG-HSA-105, CNG-HSA-106, CNG-HSA-108, CNG-HSA-109,CNG-HSA-113, CNG-HSA-116, CNG-HSA-117, or CNG-HSA-120 has a meltingtemperature greater than or equal to 60° C., as measured by differentialscanning fluorimetry. In certain embodiments, the antigen-binding sitederived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-106, orCNG-HSA-120 has a melting temperature greater than or equal to 65° C.,as measured by differential scanning fluorimetry.

The present disclosure also provides an antigen-binding site thatcompetes for binding serum (e.g., human serum albumin) and/or competesfor binding protein A with an antibody or antigen-binding sitecomprising the VH sequence provided in Table 1.

II. Multi-Specific Binding Proteins

In one aspect, the present disclosure provides a multi-specific bindingprotein that comprises a domain that binds a target molecule (e.g., atarget protein expressed on a target cell) and an antigen-binding sitedisclosed in section I above titled “Anti-Serum Albumin Antibodies.” Incertain embodiments, the multi-specific binding protein comprises afirst domain (e.g., a first antigen-binding site) that binds a firsttarget protein expressed on a target cell e.g.; and/or a second domain(e.g., a second antigen-binding site) that binds a second target proteinexpressed on an immune effector cell e.g.; and a third domain (e.g., athird antigen-binding site) that binds serum albumin (e.g., HSA),wherein the third domain comprises an antigen-binding site disclosed insection I above titled “Anti-Serum Albumin Antibodies.” The first targetcan be a molecule (e.g., protein) expressed on a target cell that isdesirable to clear (e.g., a cancer cell or a cell in a tumormicroenvironment), such as CD19, HER2, BCMA, CD33, or EGFR. The secondtarget can be a molecule (e.g., protein) expressed on an immune effectorcell (e.g., T cell or NK cell), such as CD3 (e.g., CD3ε (epsilon), CD3δ(delta), and/or CD3γ (gamma)), 4-1BB, NKG2D, or NKp30. It iscontemplated that multi-specific binding proteins binding such first andsecond targets can facilitate clearance of the cells that express thefirst target.

In certain embodiments, the first, second, and third domains comprise afirst antigen-binding site, a second antigen-binding site, and a thirdantigen-binding site, respectively. Each of the antigen-binding sites ofthe multi-specific binding protein can take various forms, such assingle-chain variable fragment (scFv), Fab fragment, or single domainantibody (sdAb). In certain embodiments, the first antigen-binding sitecomprises an scFv. In certain embodiments, the second antigen-bindingsite comprises an scFv. In certain embodiments, the thirdantigen-binding site comprises an sdAb.

Alternatively, it is also contemplated that one or more of the bindingdomains may not comprise an antigen-binding site. For example, U.S.Patent Application Publication No. US20130316952A1 discloses apolypeptide that binds serum albumin having the amino acid sequence ofLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA (SEQ ID NO: 282).Additional exemplary polypeptides that bind HSA are described in Denniset al. (2002) J. Biol. Chem., 277: 35035-43; Jacobs et al. (2015)Protein Eng. Des. Sel., 28: 385-93; and Zorzi et al. (2017) Nat.Commun., 8: 16092.

In certain embodiments, the multi-specific binding protein furthercomprises an antibody Fc region. The presence of an Fc region mayincrease the serum half-life of the multi-specific binding protein.Depending on the specific Fc subtype and variant used, the Fc region mayalso alter the activity (e.g., cytotoxic activity) of the multi-specificbinding protein.

In other embodiments, the multi-specific binding protein does notcomprise an antibody Fc region. The absence of Fc contributes to asmaller size of the multi-specific binding protein, which can exhibitimproved tissue penetration and pharmacokinetic properties. In certainembodiments, the multi-specific binding proteins consists of or consistsessentially of the first, second, and third antigen-binding sites andthe linkers between them. In certain embodiments, the multi-specificbinding proteins consists essentially of the first, second, and thirdantigen-binding sites.

In certain embodiments, the multi-specific binding protein binds thefirst target protein, the second target protein, and/or serum albuminmonovalently. The exclusion of additional binding domains reduces therisk of non-specific immune cell activation and decreases the size ofthe multi-specific binding protein.

A. First Antigen-Binding Site

In certain embodiments, the first antigen-binding site of themulti-specific binding protein binds CD19 (e.g., human CD19). In certainembodiments, the first antigen-binding site of the multi-specificbinding protein binds FLT3 (e.g., human FLT3).

The first antigen-binding site that binds CD19 can be derived from, forexample, MT-103 (a single-chain bispecific CD19/CD3 antibody; see,Hoffman et al. (2005) Int. J. Cancer, 115: 98-104; Schlereth et al.(2006) Cancer Immunol. Immunother. 55: 503-14), a CD19/CD16 diabody(see, Schlenzka et al. (2004) Anti-cancer Drugs 15: 915-19; Kipriyanovet al. (2002) J. Immunol. 169: 137-44), BU12-saporin (see, Flavell etal. (1995) Br. J. Cancer 72: 1373-79), and anti-CD19-idarubicin (see,Rowland et al. (1993) Cancer Immunol. Immunother. 55: 503-14).Additional exemplary antigen-binding sites that bind CD19, from whichthe instant first antigen-binding site may be derived, are disclosed inU.S. Patent Application Publication Nos. US20170174786A1,US20090042291A1, US20160046730A1, US20070154473A1, US20090142349A1,US20180142018A1, US20090136526A1, US20060257398A1, and US20180230225A1,and PCT Publication No. WO2019057100A1. For example, in certainembodiments, the first antigen-binding site that binds CD19 is derivedfrom an antibody listed in Table 2.

TABLE 2 Sequences of Exemplary Antibodies That Bind CD19 AntibodyVH and HCDRs VL and LCDRs CNG- QVQLVQSGAEVKKPGASVKVSCKASGDIVMTQTPLSLSVTPGQPASISCKSS CD19-1 YTFTDYIMHWVRQAPGQGLEWMGYINQSLETSTGTTYLNWYLQKPGQSPQ PYNDGSKYTEKFQGRVTMTSDTSISTALLIYRVSKRFSGVPDRFSGSGSGTD YMELSRLRSDDTAVYYCARGTYYYGPFTLKISRVEAEDVGVYYCLQLLEDP QLFDYWGQGTTVTVSS (SEQ ID NO:YTFGQGTKLEIK (SEQ ID NO: 13) 20) LCDR1: KSSQSLETSTGTTYLN (SEQHCDR1*: YTFTDYIMH (SEQ ID NO: 22) ID NO: 18) HCDR1: DYIMH (SEQ ID NO: 4)LCDR2: RVSKRFS (SEQ ID NO: 19) HCDR2: YINPYNDGSKYTEKFQG (SEQLCDR3: LQLLEDPYT (SEQ ID NO: ID NO: 23) 10)HCDR3*: ARGTYYYGPQLFDY (SEQ ID NO: 16) HCDR3: GTYYYGPQLFDY (SEQ ID NO:17) CNG- QVQLQESGPGLVKPSQTLSLTCTVSGGS EIVLTQSPATLSLSPGERATLSCSAS CD19-7ISTSGMGVGWIRQHPGKGLEWIGHIWW SSVSYMHWYQQKPGQAPRLLIYDTDDDKRYNPALKSRVTISVDTSKNQFSL SKLASGIPARFSGSGSGTDFTLTISSLKLSSVTAADTAVYYCARMELWSYYFD EPEDFAVYYCFQGSVYPFTFGQGTYWGQGTLVTVSS (SEQ ID NO: 90) KLEIK (SEQ ID NO: 74)HCDR1*: GSISTSGMGVG (SEQ ID NO: LCDR1: SASSSVSYMH (SEQ ID NO: 92) 80)HCDR1: TSGMGVG (SEQ ID NO: 93) LCDR2: DTSKLAS (SEQ ID NO: 70)HCDR2: HIWWDDDKRYNPALKS (SEQ LCDR3: FQGSVYPFT (SEQ ID NO: ID NO: 87) 71)HCDR3*: ARMELWSYYFDY (SEQ ID NO: 67) HCDR3: MELWSYYFDY (SEQ ID NO: 68)Xencor EVQLVESGGGLVKPGGSLKLSCAASGY DIVMTQSPATLSLSPGERATLSCRSS mAbTFTSYVMHWVRQAPGKGLEWIGYINP KSLQNVNGNTYLYWFQQKPGQSPYNDGTKYNEKFQGRVTISSDKSISTAY QLLIYRMSNLNSGVPDRFSGSGSGTMELSSLRSEDTAMYYCARGTYYYGTR EFTLTISSLEPEDFAVYYCMQHLEYVFDYWGQGTLVTVSS (SEQ ID NO: 197) PITFGAGTKLEIK (SEQ ID NO: 198)HCDR1: SYVMH (SEQ ID NO: 199) LCDR1: RSSKSLQNVNGNTYLYHCDR2: WIGYINPYNDGTKY (SEQ ID (SEQ ID NO: 202) NO: 200)LCDR2: RMSNLNS (SEQ ID NO: 203) HCDR3: GTYYYGTRVFDY (SEQ ID NO:LCDR3: MQHLEYPIT (SEQ ID NO: 201) 204) AbbvieQVQLQQSGAELVRPGSSVKISCKASGY DILLTQTPASLAVSLGQRATISCKAS mAbAFSSYWMNWVKQRPGQGLEWIGQIWP QSVDYDGDSYLNWYQQIPGQPPKLGDGDTNYNGKFKGKATLTADESSSTA LIYDASNLVSGIPPRFSGSGSGTDFTYMQLSSLASEDSAVYFCARRETTTVGR LNIHPVEKVDAATYHCQQSTEDPWYYYAMDYWGQGTSVTVSS (SEQ ID TFGGGTKLEIK (SEQ ID NO: 206) NO: 205)LCDR1: KASQSVDYDGDSYLN HCDR1: SYWMN (SEQ ID NO: 207) (SEQ ID NO: 210)HCDR2: QIWPGDGDTNYNGKFKG (SEQ LCDR2: DASNLVS (SEQ ID NO: 211)ID NO: 208) LCDR3: QQSTEDPWT (SEQ ID NO: HCDR3: RETTTVGRYYYAMDY (SEQ ID212) NO: 209) Immunomedics QVQLQQSGAEVKKPGSSVKVSCKASGDIQLTQSPSSLSASVGDRVTITCKAS mAb YAFSSYWMNWVRQRPGQGLEWIGQIWQSVDYDGDSYLNWYQQIPGKAPKL PGDGDTNYNGKFKGRATITADESTNTALIYDASNLVSGIPPRFSGSGSGTDYT YMELSSLRSEDTAFYSCARRETTTVGRFTISSLQPEDIATYHCQQSTEDPWTF YYYAMDYWGQGTTVTVSS (SEQ IDGGGTKLQIKR (SEQ ID NO: 214) NO: 213) LCDR1: KASQSVDYDGDSYLNHCDR1: SYWMN (SEQ ID NO: 29) (SEQ ID NO: 217)HCDR2: QIWPGDGDTNYNGKFKG (SEQ LCDR2: DASNLVS (SEQ ID NO: 218)ID NO: 215) LCDR3: QQSTEDPWT (SEQ ID NO: HCDR3: RETTTVGRYYYAMDY (SEQ ID219) NO: 216) Merck QVQLEQPGAEVVKPGASVKVSCKTSG QIVLTQSPATLSASPGEKATMTCSAmAb YTFTSNWMHWVKQTPGKGLEWIGEID SSGVNYMHWYQQKPGTSPKRWIYPSDSYTNYNQKFDGKAKLTVDKSSSTA DTDKTASGVPARFSGSGSGTSYSLTYMEVSDLTAEDSATYYCARGSNPYYY ISSMEAEDAATYYCHQRGSYTFGGAMDYWGQGTSVTVSS (SEQ ID NO: GTKLEIK (SEQ ID NO: 221) 220)LCDR1: SASSGVNYMH (SEQ ID NO: HCDR1: SNWMH (SEQ ID NO: 222) 225)HCDR2: EIDPSDSYTN (SEQ ID NO: 223) LCDR2: DTDKTAS (SEQ ID NO: 226)HCDR3: GSNPYYYAMDY (SEQ ID NO: LCDR3: HQRGSYT (SEQ ID NO: 227) 224)Medarex EVQLVQSGAEVKKPGESLKISCKGSGY AIQLTQSPSSLSASVGDRVTITCRAS mAbSFSSSWIGWVRQMPGKGLEWMGIIYPD QGISSALAWYQQKPGKAPKLLIYD 21D4aDSDTRYSPSFQGQVTISADKSIRTAYLQ ASSLESGVPSRFSGSGSGTDFTLTISWSSLKASDTAMYYCARHVTMIWGVII SLQPEDFATYYCQQFNSYPFTFGPGDFWGQGTLVTVSS (SEQ ID NO: 228) TKVDIK (SEQ ID NO: 229)HCDR1: SSWIG (SEQ ID NO: 230) LCDR1: RASQGISSALA (SEQ ID NO:HCDR2: IIYPDDSDTRYSPSFQG (SEQ ID 233) NO: 231)LCDR2: DASSLES (SEQ ID NO: 234) HCDR3: HVTMIWGVIIDF (SEQ ID NO:LCDR3: QQFNSYPFT (SEQ ID NO: 232) 235) MedarexEVQLVQSGAEVKKPGESLKISCKGSGY AIQLTQSPSSLSASVGDRVTITCRAS mAbSFSSSWIGWVRQMPGKGLEWMGIIYPD QGISSALAWYQQKPGKAPKLLIYD 21D4DSDTRYSPSFQGQVTISADKSIRTAYLQ ASSLESGVPSRFSGSGSGTDFTLTISWSSLKASDTAMYYCARHVTMIWGVII SLQPEDFATYYCQQFNSYPYTFGQDFWGQGTLVTVSS (SEQ ID NO: 236) GTKLEIK (SEQ ID NO: 237)HCDR1: SSWIG (SEQ ID NO: 238) LCDR1: RASQGISSALA (SEQ ID NO:HCDR2: IIYPDDSDTRYSPSFQG (SEQ ID 241) NO: 239)LCDR2: DASSLES (SEQ ID NO: 242) HCDR3: HVTMIWGVIIDF (SEQ ID NO:LCDR3: QQFNSYPYT (SEQ ID NO: 240) 243) MedarexQVQLVQSGAEVKKPGSSVKVSCKDSG EIVLTQSPGTLSLSPGERATLSCRAS mAbGTFSSYAISWVRQAPGQGLEWMGGIIPI QSVSSSYLAWYQQKPGQAPRLLIY 47G4FGTTNYAQQFQGRVTITADESTSTAYM GASSRATGIPDRFSGSGSGTDFTLTIELSSLRSEDTAVYYCAREAVAADWLDP SRLEPEDFAVYYCQQYGSSRFTFGPWGQGTLVTVSS (SEQ ID NO: 244) GTKVDIK (SEQ ID NO: 245)HCDR1: SYAIS (SEQ ID NO: 246) LCDR1: RASQSVSSSYLA (SEQ IDHCDR2: GIIPIFGTTNYAQQFQG (SEQ ID NO: 249) NO: 247)LCDR2: GASSRAT (SEQ ID NO: 250) HCDR3: EAVAADWLDP (SEQ ID NO:LCDR3: QQYGSSRFT (SEQ ID NO: 248) 251) MedarexEVQLVQSGAEVKKPGESLKISCKGSGY AIQLTQSPSSLSASVGDRVTITCRAS mAb 27F3SFTSYWIAWVRQMPGKGLEWMGIIYPG QGISSALAWYQQKPGKAPKLLIYDDSDTRYSPSFQGQVTISADKSISTAYLQ ASSLESGVPSRFSGSGSGTDFTLTISWSSLKASDTAMYYCARQGYSSGWDSY SLQPEDFATYYCQQFNSYPYTFGQYGMGVWGQGTTVTVSS (SEQ ID NO: GTKLEIK (SEQ ID NO: 253) 252)LCDR1: RASQGISSALA (SEQ ID NO: HCDR1: SYWIA (SEQ ID NO: 254) 257)HCDR2: IIYPGDSDTRYSPSFQG (SEQ ID LCDR2: DASSLES (SEQ ID NO: 258)NO: 255) LCDR3: QQFNSYPYT (SEQ ID NO: HCDR3: QGYSSGWDSYYGMGV (SEQ 259)ID NO: 256) Medarex QVQLVQSGAEVKKPGSSVKVSCKASG DIQMTQSPSSLSASVGDRVTITCRAmAb GTFSSYTINWVRQAPGQGLEWMGGIIPI SQGISSWLAWYQQKPEKAPKSLIY 3C10FGIPNYAQKFQGRVTITADESTNTAYM AASSLQSGVPSRFSGSGSGTDFTLTIELSSLRAEDTAVYYCARASGGSADYSY SSLQPEDFATYYCQQYKRYPYTFGGMDVWGQGTAVTVSS (SEQ ID NO: QGTKLEIK (SEQ ID NO: 261) 260)LCDR1: RASQGISSWLA (SEQ ID HCDR1: SYTIN (SEQ ID NO: 262) NO: 265)HCDR2: GIIPIFGIPNYAQKFQG (SEQ ID LCDR2: AASSLQS (SEQ ID NO: 266)NO: 263) LCDR3: QQYKRYPYT (SEQ ID NO: HCDR3: ASGGSADYSYGMDV (SEQ ID 267)NO: 264) Medarex EVQLVQSGAEVKKPGESLNISCKGSGY AIQLTQSPSSLSASVGDRVTITCRASmAb 5G7 SFTSYWIGWVRQMPGKGLEWMGIIYPG QGISSALAWYQQKPGKAPKLLIYDDSDTRYSPSFQGQVTISADKSINTAYLQ ASSLESGVPSRFSGSGSGTDFTLTISWSSLKASDTAMYYCARGVSMIWGVIM SLQPEDFATYYCQQFNSYPWTFGQDVWGQGTTVTVSS (SEQ ID NO: 268) GTKVEIK (SEQ ID NO: 269)HCDR1: SYWIG (SEQ ID NO: 270) LCDR1: RASQGISSALA (SEQ ID NO:HCDR2: IIYPGDSDTRYSPSFQG (SEQ ID 273) NO: 271)LCDR2: DASSLES (SEQ ID NO: 274) HCDR3: GVSMIWGVIMDV (SEQ ID NO:LCDR3: QQFNSYPWT (SEQ ID NO: 272) 275) MedarexEVQLVQSGAEVKKPGESLQISCKGSGY AIQLTQSPSSLSASVGDRVTITCRAS mAb 13F1TFTNYWIAWVRQMPGKGLEWMGIIYP QGISSALAWYQQKPGKAPKLLIYDGDSDTRYSPSFQGQVTISADKSISTAYL ASSLESGVPSRFSGSGSGTDFTLTISQWSGLKASDTAMYYCARQGYSSGWR SLQPEDFATYYCQQFNSYPHTFGQSYYGMGVWGQGTTVTVSS (SEQ ID GTKLEIK (SEQ ID NO: 277) NO: 276)LCDR1: RASQGISSALA (SEQ ID NO: HCDR1: NYWIA (SEQ ID NO: 278) 281)HCDR2: IIYPGDSDTRYSPSFQG (SEQ ID LCDR2: DASSLES (SEQ ID NO: 282)NO: 279) LCDR3: QQFNSYPHT (SEQ ID NO: HCDR3: QGYSSGWRSYYGMGV (SEQ 283)ID NO: 280) Medarex EVQLVQSGAEVKKPGESLQISCKGSGYAIQLTQSPSSLSASVGDRVTITCRAS mAb 46E8 TFTNYWIAWVRQMPGKGLEWMGIIYPQGISSALAWYQQKPGKAPKLLIYD GDSDTRYSPSFQGQVTISADKSISTAYLASSLESGVPSRFSGSGSGTDFTLTIS QWSGLKASDTAMYYCARQGYSSGWRSLQPEDFATYYCQQFNSYPHTFGQ SYYGMGVWGQGTTVTVSS (SEQ IDGTKLEIK (SEQ ID NO: 285) NO: 284) LCDR1: RASQGISSALA (SEQ ID NO:HCDR1: NYWIA (SEQ ID NO: 286) 289) HCDR2: IIYPGDSDTRYSPSFQG (SEQ IDLCDR2: DASSLES (SEQ ID NO: 290) NO: 287) LCDR3: QQFNSYPHT (SEQ ID NO:HCDR3: QGYSSGWRSYYGMGV (SEQ 291) ID NO: 288) NovimmuneEVQLVQSGAEVKKPGESLKISCKGSGY DIQMTQSPSSLSASVGDRVTITCRA mAbSFTSYWIGWVRQMPGKGLEWMGIIYPG SQSISSYLNWYQQKPGKAPKLLIYADSDTRYSPSFQGQVTISADKSISTAYLQ ASSLQSGVPSRFSGSGSGTDFTLTISWSSLKASDTAMYYCARGVSGIYNLHG SLQPEDFATYYCQQGRFGSPFTFGQFDIWGQGTLVTVSS (SEQ ID NO: 315) GTKVEIK (SEQ ID NO: 316)HCDR1: GYSFTSYW (SEQ ID NO: 317) LCDR1: QSISSY (SEQ ID NO: 320)HCDR2: IYPGDSDT (SEQ ID NO: 318) LCDR2: AAS (SEQ ID NO: 321)HCDR3: ARGVSGIYNLHGFDI (SEQ ID LCDR3: QQGRFGSPFT (SEQ ID NO: NO: 319)322) Eureka QVQLVETGGGLVQPGGSLRLSCAASGF QTVVTQEPSVSAAPGQKVTISCSGS mAb-1TFSSYAMSWVRQAPGKGLEWVSAISGS SSNIGNNYVSWYQQLPGTAPKLLIYGGSTYYADSVKGRFTISRDNSKNTLYL DNNKRPSGIPDRFSGSKSGTSATLGIQMNSLRAEDTAVYYCARYYYSRLDY TGLQTGDEADYYCGTWDSSLSAGVWGQGTLVTVSS (SEQ ID NO: 323) FGTGTKLTVLGSR (SEQ ID NO: 324) EurekaQVQLVESGGGLVQPGGSLRLSCAASGF QSVLTQPPSVSAAPGQKVTISCSGSS mAb-2TFSSYAMSWVRQAPGKGLEWVSGISAS SNIGNNYVSWYRQLPGTAPKLLIYEGGSTYYADSVKGRFTISRDNSKNTLYL NNKRPSGIPDRFSGSKSGTSATLGITQMNSLRAEDTAVYYCARYYLSQIDSW GLQTGDEADYYCGTWDSSLRAGVGQGTLVTVSS (SEQ ID NO: 325) FGTGTKVTVL (SEQ ID NO: 326) EurekaEVQLVQSGAEVKKPGATVKISCKVSGY QSVLTQPPSASGTPGQRVTISCSGSS mAb-3TFTDYYMHWVQQAPGKGLEWMGLVD SNIGSNTVNWYQQLPGTAPKLLIYSPEDGETIYAEKFQGRVTITADTSTDTAY NNQRPSGVPDRFSGSKSGTSASLAIMELSSLRSEDTAVYYCATGIYSRPLGY SGLQSEDEADYYCAAWDDSLNGHWGQGTLVTVSS (SEQ ID NO: 327) VVFGGGTKLTVL (SEQ ID NO: 328) EurekaEVQLVETGGGLVQPGGSLRLSCAASGF SYVLTQPPSASGTPGQRVTISCSGSS mAb-4TFSSYAMSWVRQAPGKGLEWVSAISGS SNIGSHTVNWYQQLPGTAPKLLIYSGGSTYYADSVKGRFTISRDNSKNTLYL NNQRPSGVPDRFSGSKSGTSASLAIQMNSLRAEDTAVYYCARSDGKHFWQ SGLQSEDEADYYCAAWDDSLNGYQYDAWGQGTLVTVSS (SEQ ID NO: VFGTGTKVTVL (SEQ ID NO: 330) 329) EurekaEVQLVESGGGLVQPGGSLRLSCAASGF DIQLTQSPSSLSAYVGDRVTITCRAS mAb-5TVS SNYMS WVRQAPGKGLEWVS AISG QGITNSLAWYQQKPGKAPKLLLHASGGSTYYADSVKGRFTISRDNSKNTLY ASRLESGVPSRFSGSGFGTDFTLTISLQMNSLRAEDTAVYYCARMNIDYWG SLQPEDFAVYYCQHYLGTPYSFGQQGTLVTVSS (SEQ ID NO: 331) GTKVEIK (SEQ ID NO: 332) EurekaEVQLVQSGAEVKRPGESLTISCKGSEYS EIVLTQSPSSLSASVGDRVTISCRAS mAb-6FASYWITWVRQMPGKGLEWMGRIDPS QSVSRFLNWYQQKPGKAPKLLIYGDSYTNYSPSFQGHVTISADKSISTAYLQ VSTLERGVPSRFSGSGSGTDFTLTISWS SLKASDTAIYYCARPFQYDYGGYSD SLQPEDFATYYCQESYIIPLTFGGGTAFDIWGQGTMVTVSS (SEQ ID NO: 333) KLEIK (SEQ ID NO: 334) EurekaQMQLVQSGAEVKKAGSSVKVSCETSG EIVMTQSPLSLSVTPGEPASISCRSS mAb-7GTFSSSSVNWVRQAPGQGLEWMGGIIPI QSLLDSNGFNSLDWYLQKPGQSPQVGTPNYAQKFQDRVTITAVESTFTAYM LLIHLGSDRASGVPDRFSGSGSGTDELSGLRSEDTAVYYCARGGYRDYMDV FTLKISRVEAEDVGIYYCMQSLQIPTWGRGTTVTVSS (SEQ ID NO: 335) FGQGTKVEIK (SEQ ID NO: 336) EurekaEVQLVESGGGLIQPGGSLRLSCAASGFT SYELTQPPSASGTPGQRVTISCSGSS mAb-8VSSNYMSWVRQAPGKGLEWVSVIYSG SNIGSNYVYWYQQLPGTAPKLLIYRGSTYYADSVKGRFTISRDNSKNTLYLQ NNQRPSGVPDRFSGSKSGTSASLAIMNSLRAEDTAVYYCARGGFGAEFDYW SGLRSEDEADYYCAAWDDSLSGYVGQGTLVTVSS (SEQ ID NO: 337) FGTGTKVTVL (SEQ ID NO: 338) EurekaEVQLVESGGGLIQPGGSLRLSCAASGFT SYVLTQPPSVSVSPGQTASITCSGD mAb-9VSSNYMSWVRQAPGKGLEWVSVIYSG KLGDKYASWYQQKPGQSPVLVIYQGSTYYADSVKGRFTISRDNSKNTLYLQ DNKRPSGIPERFSGSNSGNTATLTISMNSLRAEDTAVYYCARGGISDDYYGS GTQAMDEADYYCQAWDSSTEDVFGSYDNWGQGTLVTVSS (SEQ ID NO: GPGTKVTVL (SEQ ID NO: 340) 339) EurekaEVQLVESGGGLVQPGGSLRLSCAASGF DIQLTQSPSSLSASVGDRVTITCRAS mAb-10TVSSNYMSWVRQAPGKGLEWVSVIYS QSISSYLNWYQQKPGKAPKLLIYAAGGSTYYADSVKGRFTISRDNSKNTLYL SSLQSGVPSRFSGSGSGTDFTLTISSQMNSLRAEDTAVYYCARERGMGYAFD LQPEDFATYYCQQSYSTPFTFGGGTIWGQGTMVTVSS (SEQ ID NO: 341) KVEIK (SEQ ID NO: 342) EurekaQLQLQESGPGLVKPSETLSLTCSVSGVS DIQMTQSPSSLSASVGDRVTITCRA mAb-11MSENYWSWIRQPPGKRLEWIGCAHYT SQGIGSYLAWYQQKPGKAPKLLIYPGDTHYNPSLKGRVTISLDTSMNQFSLRL ASTLQSGVPSRFSGSGSGTEFTLTISNSVTAADTAVYYCASYHPFNYWGQGT SLQPEDFATYYCQQLNSLFGQGTRLLVTVSS (SEQ ID NO: 343) EIK (SEQ ID NO: 344) EurekaEVQLVQSGAEVRRPGATVKISCKVSGY QAVLTQPPSASGTPGQRVTISCSGSS mAb-12TFNDFYLHWVRQAPGKGLEWMGRIDP SNIGTKTVNWYQVLPGTAPKLLIYSEDGKTRYAEKFQGRLTITADTSTDTLY NYRRPSGVPDRFSGSKSGTSASLAISMQLGGLTSDDTAVYYCTTDWGYSSSL GLQSDDEADYYCALWDDSLDGYVREEDIWYDCWGQGTLVTVSS (SEQ ID FGTGTKVTVL (SEQ ID NO: 346) NO: 345) EurekaEVQLVQSGAEVKKPGSSVKVSCKASGG SYELTQPPSVSVAPGKTARITCGGN mAb-13TFSSYAISWVRQAPGQGLEWMGGIIPIF NIGSKSVHWYQQKPGQAPVLVIYYGTANYAQKFQGRVTITADESTSTAYME DSDRPSGIPERFSGSNSGNTATLTISLSSLRSEDTAVYYCARDYGYGD YGD A RVEAGDEADYYCQVWDSSSDHYVFDIWGQGTMVTVSS (SEQ ID NO: 347) FGTGTKVTVL (SEQ ID NO: 348) EurekaEVQLVQSGAEVKKPGESLKISCKGSGY SYVLTQPPSVSVAPGKTARITCGGN mAb-14SFTSYWIGWVRQMPGKGLEWMGIIYPG NIGSKSVHWYQQRPGQAPVLVVYDDSDTRYSPSFQGQVTISADKSISTAYLQ DSDRPSGIPERFSGSNSGNTATLTISWSSLKASDTAMYYCARVVGTIYSMQY RVEAGDEADYSCQVWDSSSDHYVDVWGQGTLVTVSS (SEQ ID NO: 349) FGPGTKVTVL (SEQ ID NO: 350) EurekaEVQLVQSGAEVKKPGESLKISCKGSGY LPVLTQPPSVSVAPGKTARITCGGN mAb-15SFTSYWIGWVRQMPGKGLEWMGIIYPG NIGSKSVHWYQQKPGQAPVLVVYDSDTRYSPSFQGQVTISADKSISTAYLQ DDSDRPSGIPERFSGSNSGNTATLTIWSSLKASDTAMYYCARQVWGWQGG SRVEAGDEADYYCQVWDSSSDYVMYPRSNWWYNMDSWGQGTLVTVSS VFGGGTKLTVL (SEQ ID NO: 352) (SEQ ID NO: 351)Eureka EVQLVQSGAEVKKPGESLKISCKGSGY QAVLTQPPSVSEAPRQRVTISCSGSS mAb-16SFTSYWIGWVRQMPGKGLEWMGIIYPG SNVGNNAVNWYQQVPGKAPKLLIDSDTRYSPSFQGQVTISADKSISTAYLQ YYDDLLSSGVSDRFSGSKSGTSASLWSSLKASDTAMYYCARWSSTWDSMY AISGLQSEDEADYYCAAWDDSLNGMDYWGQGTLVTVSS (SEQ ID NO: 353) PVFGGGTKLTVL (SEQ ID NO: 354) EurekaEVQLVQSGAEVKKPGESLRISCKGSGY QPVLTQPPSVSVAPGKTARITCGGN mAb-17SFTSYWIGWVRQMPGKGLEWMGIIYPG NIGSESVHWYQQKPGQAPMVVIYYDSDTRYSPSFQGQVTISADKSISTAYLQ DSNRPSGIPERFSGSNSGNTATLTVSWSSLKASDTAMYYCARVTYSMDSYYF RVEAEDEADYYCQVWNSSSDHRGDSWGQGTLVTVSS (SEQ ID NO: 355) VFGGGTKLTV (SEQ ID NO: 356) WuXiEVQLQQSGPELVKPGASVKMSCKASG DAVMTQTPLSLPVSLGDQASISCRS WBP7011-YTFTNYVIHWVKQKPGQGLEWIGYFNP SQSLENSNGNTYLNWYLQKPGQSP 4.34.11YNDGTEYNEKFKAKATLTSDKSSSTAY QLLIYRVSNRFSGVLDRFSGSGSGTMELSSLTSEDSAVYYCAKGPYYYGSSP DFTLKISRVEAEDLGVYFCLQVTHVFDYWGQGTTLTVSS (SEQ ID NO: 357) PYTFGGGTKLEIK (SEQ ID NO: 358)HCDR1: GYTFTNYVIH (SEQ ID NO: 359) LCDR1: RSSQSLENSNGNTYLNHCDR2: YFNPYNDGTEYNEKFKA (SEQ (SEQ ID NO: 362) ID NO: 360)LCDR2: RVSNRFS (SEQ ID NO: 363) HCDR3: GPYYYGSSPFDY (SEQ ID NO:LCDR3: RVSNRFS (SEQ ID NO: 364) 361) WuXi QVQLQQSGAELVRPGSSVKISCKASGYDIQMTQTTSSLSASLGDRVTISCRAS WBP7011- AFSTYWMNWVKQRPGQGLEWIGQIYPQDISNYLNWYQQKPDGTVKLLIYY 4.87.6 GDDDTKYNGKFKGKASLTADKSSSTATSRLHSGVPARFSGSGSGTDYSLTIS YMQLISLTSEDSAVYFCARRYFRYDYWNLEQEDIATYFCHQGNTLPLTFGAG YSDVWGAGTTVTVTS (SEQ ID NO: 365)TKLELK (SEQ ID NO: 366) HCDR1: GYAFSTYWMN (SEQ ID NO:LCDR1: RASQDISNYLN (SEQ ID 367) NO: 370) HCDR2: QIYPGDDDTKYNGKFKG (SEQLCDR2: YTSRLHS (SEQ ID NO: 371) ID NO: 368) LCDR3: HQGNTLPLT (SEQ ID NO:HCDR3: RYFRYDYWYSDV (SEQ ID NO: 372) 369) WuXiEIQLQQSGPELVKPGASVKVSCKASGY QIVLTQSPAIMSASLGEEITLTCSAS WBP7011_AFTSYNMYWVKQSHGKSLEWIGYIDP STVNYMHWYQQKSGTSPKLLIYST 4.155.8YNGDTTYNQKFKGKATLTVDKSSSTA SNLASGVPSRFSGSGSGTFYSLTIRSYMHLNSLTSEDSAVYYCLTTAYAMDY VEAEDAADYYCHQWSSYPYTFGGWGQGTSVTVSS (SEQ ID NO: 373) GTKLEIK (SEQ ID NO: 374)HCDR1: GYAFTSYNMY (SEQ ID NO: LCDR1: SASSTVNYMH (SEQ ID NO: 375) 378)HCDR2: YIDPYNGDTTYNQKFKG (SEQ LCDR2: STSNLAS (SEQ ID NO: 379)ID NO: 376) LCDR3: HQWSSYPYT (SEQ ID NO: HCDR3: TAYAMDY (SEQ ID NO: 377)380) WuXi QVQLVQSGAEVKKPGSSVKVSCKASG DIVMTQTPLSLPVTPGEPASISCRSS WBP7011-YTFTDYVIHWVRQAPGQGLEWMGYFN QSLENSNHNTYINWYLQKPGQSPQ 4.34.11-PYNDGTEYNEKFKARVTITADKSTSTA LLIYRVSKRFSGVPDRFSGSGSGTD z1-m5YMELSSLRSEDTAVYYCARGPYYYGSS FTLKISRVEAEDVGVYYCHQVTHVPFDYWGQGTTVTVSS (SEQ ID NO: 381) PYTFGQGTKLEIK (SEQ ID NO: 382)HCDR1: GYTFTNYVIH (SEQ ID NO: 383) LCDR1: RSSQSLENSNHNTYIN (SEQHCDR2: YFNPYNDGTEYNEKFKA (SEQ ID NO: 386) ID NO: 384)LCDR2: RVSKRFS (SEQ ID NO: 19) HCDR3: GPYYYGSSPFDY (SEQ ID NO:LCDR3: HQVTHVPYT (SEQ ID NO: 385) 387) WuXi QVQLVQSGAEVKKPGASVKVSCKASGDIQMTQSPSSLSASVGDRVTITCRA WBP7011- YAFSTYWMNWVRQAPGQGLEWMGQISQDISNYLNWYQQKPGKVPKLLIY 4.87.6- YPGDDDTKYSGKFKGRVTITADKSTSTYTSRLHSGVPSRFSGSGSGTDFTLTI z1(N-S) AYMELSSLRSEDTAVYYCARRYFRYDSSLQPEDVATYYCHQGNTLPLTFG YWYSDVWGQGTTVTVSS (SEQ ID NO:QGTKLEIK (SEQ ID NO: 389) 388) LCDR1: RASQDISNYLN (SEQ IDHCDR1: GYAFSTYWMN (SEQ ID NO: NO: 393) 390)LCDR2: YTSRLHS (SEQ ID NO: 394) HCDR2: QIYPGDDDTKYSGKFKG (SEQLCDR3: HQGNTLPLT (SEQ ID NO: ID NO: 391) 395)HCDR3: RYFRYDYWYSDV (SEQ ID NO: 392) WuXi QMQLVQSGPEVKKPGTSVKVSCKASGDIQLTQSPSFLSASVGDRVTITCSAS WBP7011_ YAFTSYNMYWVRQARGQRLEWIGYIDSTVNYMHWYQQKPGKAPKLLIYST 4.155.8- PYNADTTYNQKFKGRVTITRDMSTSTASNLASGVPSRFSGSGSGTEFTLTISS z1-P15 YMELS SLRSEDTAVYYCLTTAYAMD YLQPEDFATYYCHQWSSYPYTFGQG WGQGTLVTVSS (SEQ ID NO: 396)TKLEIK (SEQ ID NO: 397) HCDR1: GYAFTSYNMY (SEQ ID NO:LCDR1: SASSTVNYMH (SEQ ID NO: 398) 401) HCDR2: YIDPYNADTTYNQKFKG (SEQLCDR2: STSNLAS (SEQ ID NO: 402) ID NO: 399) LCDR3: HQWSSYPYT (SEQ ID NO:HCDR3: TAYAMDY (SEQ ID NO: 400) 403) Legend QVKLEESGGELVQPGGPLRLSCAASGNIN/A mAb FSINRMGWYRQAPGKQRAFVASITVRG ITNYADSVKGRFTISVDKSKNTIYLQMNALKPEDTAVYYCNAVSSNRDPDYWGQ GTQVTVSS (SEQ ID NO: 404)HCDR1: INRMG (SEQ ID NO: 405) HCDR2: SITVRGITNYADSVKG (SEQ ID NO: 406)HCDR3: VSSNRDPDY (SEQ ID NO: 407) Where the VL and LCDR sequences arenoted as “N/A,” the antigen-binding site is an sdAb having a VH (e.g.,VHH) only.

In certain embodiments, the first antigen-binding site comprises a VHthat comprises an amino acid sequence at least 60% (e.g., at least 70%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) identical to the VH of an antibodydisclosed in Table 2, and a VL that comprises an amino acid sequence atleast 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto the VL of the same antibody disclosed in Table 2. In certainembodiments, the antigen-binding site comprises the HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3, determined under Kabat (see Kabat et al.,(1991) Sequences of Proteins of Immunological Interest, NIH PublicationNo. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum R M et al.,(1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) TheImmunologist, 7, 132-136), or any other CDR determination method knownin the art, of the VH and/or VL sequences of an antibody disclosed inTable 2. In certain embodiments, the antigen-binding site comprises theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibodydisclosed in Table 2. In certain embodiments, the antigen-binding sitecomprises the VH and VL sequences of an antibody disclosed in Table 2.

Such antigen-binding site may take the form of scFv. In certainembodiments, the VH is positioned C-terminal to the VL. In certainembodiments, the VH is positioned N-terminal to the VL. In certainembodiments, the VH and the VL are linked by a peptide linker, forexample, a linker disclosed in subsection D below titled “Linkers.” Tostabilize the scFv, the amino acid residues at position 44 of the VH andat position 100 of the VL (under Kabat numbering) can be substituted byCys, thereby facilitating the formation of a disulfide bond between theVH and the VL. Accordingly, in certain embodiments, the VH and VLcomprise Cys at positions 100 and 44, respectively.

In other embodiments, the first antigen-binding site comprises an sdAbcomprising a VH comprising complementarity determining regions HCDR1,HCDR2, and HCDR3. In certain embodiments, the VH comprises an amino acidsequence at least 60% (e.g., at least 70%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the VH of an sdAb antibody provided in Table 2. In certainembodiments, the VH comprises the HCDR1, HCDR2, and HCDR3, determinedunder Kabat (see Kabat et al., (1991) Sequences of Proteins ofImmunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia(see, e.g., Chothia C & Lesk A M, (1987), J Mol Biol 196: 901-917),MacCallum (see MacCallum R M et al., (1996) J Mol Biol 262: 732-745),IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any otherCDR determination method known in the art, of the VH sequence of anantibody disclosed in Table 2. In certain embodiments, the VH comprisesthe HCDR1, HCDR2, and HCDR3 sequences of the antibody provided in Table2. In certain embodiments, the VH comprises the amino acid sequence ofthe VH of an sdAb provided in Table 2.

In certain embodiments, the first antigen-binding site binds CD19 with aK_(D) lower than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM,40 pM, 30 pM, 20 pM, or 10 pM. For example, in certain embodiments, thefirst antigen-binding site binds CD19 with a K_(D) about 10 pM-about 1nM, about 10 pM-about 0.9 nM, about 10 pM-about 0.8 nM, about 10pM-about 0.7 nM, about 10 pM-about 0.6 nM, about 10 pM nM-about 0.5 nM,about 10 pM-about 0.4 nM, about 10 pM-about 0.3 nM, about 10 pM-about0.2 nM, about 10 pM-about 0.1 nM, about 10 pM-about 50 pM, 0.1 nM-about10 nM, about 0.1 nM-about 9 nM, about 0.1 nM-about 8 nM, about 0.1nM-about 7 nM, about 0.1 nM-about 6 nM, about 0.1 nM-about 5 nM, about0.1 nM-about 4 nM, about 0.1 nM-about 3 nM, about 0.1 nM-about 2 nM,about 0.1 nM-about 1 nM, about 0.1 nM-about 0.5 nM, about 0.5 nM-about10 nM, about 1 nM-about 10 nM, about 2 nM-about 10 nM, about 3 nM-about10 nM, about 4 nM-about 10 nM, about 5 nM-about 10 nM, about 6 nM-about10 nM, about 7 nM-about 10 nM, about 8 nM-about 10 nM, or about 9nM-about 10 nM.

It is understood that the binding affinity to CD19 of the firstantigen-binding site alone may be different from the binding affinity ofthe same antigen-binding site in the context of the multi-specificbinding protein disclosed herein, possibly due to the conformationalrestraint from the other domains. The context-dependent binding affinityis described in subsection E below titled “Binding Affinity”

In certain embodiments, the first antigen-binding site, when present inthe form of an Fab, has a melting temperature of at least 60° C., atleast 65° C., at least 70° C., at least 75° C., or at least 80° C. Incertain embodiments, the first antigen-binding site, when present in theform of an Fab, has a melting temperature in the range of 60-85° C.,60-80° C., 60-75° C., 60-70° C., 60-65° C., 65-85° C., 65-80° C., 65-75°C., 65-70° C., 70-85° C., 70-80° C., 70-75° C., 75-85° C., 75-80° C., or80-85° C.

B. Second Antigen-Binding Site

In certain embodiments, the second antigen-binding site of themulti-specific binding protein binds CD3 (e.g., human CD3 and/or MacacaCD3). In certain embodiments, the second antigen-binding site binds CD3ε(epsilon). In certain embodiments, the second antigen-binding site bindsCD3δ (delta). In certain embodiments, the second antigen-binding sitebinds CD3γ (gamma).

Earlier BiTE® constructs bind conformational epitopes of CD3 andtypically are species-specific (see, PCT Publication No.WO2008119567A2). Improved BiTE® constructs, such as blinatumomab (alsocalled AMG 103; see, PCT Publication No. WO1999054440A1) and solitomab(also called AMG 110; see, PCT Publication No. WO2005040220A1), bindcontext-independent epitopes at the N-terminus of CD3ε chain (e.g.,amino acid residues 1-27 of human CD3ε extracellular domain) and showcross-species specificity for human, Callithrix jacchus, SaguinusOedipus, and Saimiri sciureus CD3ε chain (see id.). These constructs donot nonspecifically activate T cells to the same degree as observed withthe earlier BiTE® constructs, and are therefore believed to bear a lowerrisk of side effects (see, Brischwein et al. (2007) J. Immunother.,30(8): 798-807).

In certain embodiments, the second antigen-binding site of themulti-specific binding protein binds an epitope at the N-terminus ofCD3ε chain. In certain embodiments, the second antigen-binding sitebinds an epitope localized in amino acid residues 1-27 of human CD3εextracellular domain. This epitope or a homologous variant thereof isalso present in certain non-human primates. Accordingly, in certainembodiments, the second antigen-binding site binds CD3 in differentprimates, for example, human, new world primates (such as Callithrixjacchus, Saguinus Oedipus, or Saimiri sciureus), old world primates(such as baboons and macaques), gibbons, and non-human homininae.Callithrix jacchus and Saguinus oedipus are new world primates belongingto the family of Callitrichidae, while Saimiri sciureus is a new worldprimate belonging to the family of Cebidae. In certain embodiments, thesecond antigen-binding site binds human CD3ε and/or Macaca CD3ε. Incertain embodiments, the second antigen-binding site further bindsCallithrix jacchus, Saguinus Oedipus, and/or Saimiri sciureus CD3ε.

The second antigen-binding site that binds an extracellular epitope ofhuman and/or Macaca CD3 can be derived from, for example, muromonab-CD3(OKT3) as described in WO2008101154; otelixizumab (TRX4) as described inWO2007145941; teplizumab (MGA031) as described in WO2013040164;visilizumab (Nuvion) as described in WO2004052397; SP34 as described inWO2015181098; X35, VIT3, or BMA030 (BW264/56) as described inWO2015006749; CLB-T3/3, CRIS7, CLB-T3.4.2, WT32, 11D8, XIII-141,XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, orF101.01 as described in WO2004106381, YTH12.5 or SPv-T3b as described inWO2004106383, F1 11-409 as described in WO2012084895, TR-66 as describedin WO2013158856; UCHT-1 as described in WO2000041474; WT-31 as depictedin WO2016085889, or an antibody described in WO2008119567. For example,in certain embodiments, the second antigen-binding site that binds CD3is derived from an antibody listed in Table 3.

TABLE 3 Sequences of Exemplary Antibodies That Bind CD3 AntibodyVH and HCDRs VL and LCDRs CNG-CD3- QVQLVQSGAEVKKPGASVKVSCKASGDIVMTQSPDSLAVSLGERATINCKS 1 FNIKDYYMHWVRQAPGQRLEWMGWISQSLLNARTGKNYLAWYQQKPGQP DLENANTIYDAKFQGRVTITRDTSASTPKLLIYWASTRESGVPDRFSGSGSG AYMELSSLRSEDTAVYYCARDAYGRYTDFTLTISSLQAEDVAVYYCKQSYS FYDVWGQGTLVTVSS (SEQ ID NO:RRTFGGGTKVEIK (SEQ ID NO: 413) 412) LCDR1: KSSQSLLNARTGKNYLAHCDR1*: FNIKDYYMH (SEQ ID NO: (SEQ ID NO: 419) 414)LCDR2: WASTRES (SEQ ID NO: 420) HCDR1: DYYMH (SEQ ID NO: 415)LCDR3: KQSYSRRT (SEQ ID NO: HCDR2: WIDLENANTIYDAKFQG (SEQ 421)ID NO: 416) HCDR3*: ARDAYGRYFYDV (SEQ ID NO: 417)HCDR3: DAYGRYFYDV (SEQ ID NO: 418) scFv:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 422)scFv with Cys substitutions:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFG C GTKVEIKGGGGSGGGGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKVSCKASGF NIKDYYMHWVRQAPGQ CLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 423) CNG-CD3-QVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS 2FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNARTGKNYLAWYQQKPGQPDLENANTIYDAKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDQYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: RRTFGGGTKVEIK (SEQ ID NO: 413) 424)LCDR1: KSSQSLLNARTGKNYLA HCDR1*: FNIKDYYMH (SEQ ID NO: (SEQ ID NO: 419)414) LCDR2: WASTRES (SEQ ID NO: 420) HCDR1: DYYMH (SEQ ID NO: 415)LCDR3: KQSYSRRT (SEQ ID NO: HCDR2: WIDLENANTIYDAKFQG (SEQ 421)ID NO: 416) HCDR3*: ARDQYGRYFYDV (SEQ ID NO: 425)HCDR3: DQYGRYFYDV (SEQ ID NO: 426) scFv:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDQYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 427)scFv with Cys substitutions:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFG C GTKVEIKGGGGSGGGGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKVSCKASGF NIKDYYMHWVRQAPGQ CLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDQYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 428) CNG-CD3-QVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS 3FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNARTGKNYLAWYQQKPGQPDLEEGNTIYDAKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDAYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: LRTFGGGTKVEIK (SEQ ID NO: 430) 429)LCDR1: KSSQSLLNARTGKNYLA HCDR1*: FNIKDYYMH (SEQ ID NO: (SEQ ID NO: 419)414) LCDR2: WASTRES (SEQ ID NO: 420) HCDR1: DYYMH (SEQ ID NO: 415)LCDR3: KQSYSLRT (SEQ ID NO: HCDR2: WIDLEEGNTIYDAKFQG (SEQ 432)ID NO: 431) HCDR3*: ARDAYGRYFYDV (SEQ ID NO: 417)HCDR3: DAYGRYFYDV (SEQ ID NO: 418) scFv:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSLRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLEEGNTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 433)scFv with Cys substitutions:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSLRTFG C GTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGF NIKDYYMHWVRQAPGQ CLEWMGWIDLEEGNTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 434) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb333FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLESRTGKNYLAWYQQKPGQPDLENANTIYDAKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDVYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDLWGQGTLVTVSS (SEQ ID NO: RRTFGGGTKVEIK (SEQ ID NO: 436) 435)LCDR1: KSSQSLLNARTGKNYLA HCDR1: FNIKDYYMH (SEQ ID NO: 437)(SEQ ID NO: 440) HCDR2: WIDLENANTIYDAKFQG (SEQLCDR2: WASTRES (SEQ ID NO: 441) ID NO: 438) LCDR3: KQSYSRRT (SEQ ID NO:HCDR3: ARDVYGRYFYDL (SEQ ID NO: 442) 439) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb334FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTGKNYLAWYQQKPGQPDLENANTIYDAKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDAYGGY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: RRTFGCGTKVEIK (SEQ ID NO: 444) 443)LCDR1: KSSQSLLNARTGKNYLA HCDR1: FNIKDYYMH (SEQ ID NO: 445)(SEQ ID NO: 448) HCDR2: WIDLENANTIYDAKFQG (SEQLCDR2: WASTRES (SEQ ID NO: 449) ID NO: 446) LCDR3: KQSYSRRT (SEQ ID NO:HCDR3: ARDAYGGYFYDV (SEQ ID 450) NO: 447) AdimabEVQLLESGGGLVQPGGSLRLSCAASGF QTVVTQEPSLSVSPGGTVTLTCGSS mAb404TFDTYAMNWVRQAPGKGLEWVARIR TGAVTTSNYANWVQQTPGQAPRGSKYNNYATYYADSVKDRFTISRDDSKS LIGGTDKRAPGVPDRFSGSLLGDKATLYLQMESLRAEDTAVYYCVRHGNFG ALTITGAQAEDEADYYCALWYSNHNYAVSWFAHWGQGTLVTVSS (SEQ ID WVFGGGTKLTVL (SEQ ID NO: 452) NO: 451)LCDR1: GSSTGAVTTSNYAN (SEQ HCDR1: FTFDTYAMN (SEQ ID NO: 453) ID NO: 456)HCDR2: RIRSKYNNYATYYADSVKD LCDR2: GTDKRAP (SEQ ID NO: 457)(SEQ ID NO: 454) LCDR3: ALWYSNHWV (SEQ ID NO:HCDR3: VRHGNFGNYAVSWFAH (SEQ 458) ID NO: 455) AdimabEVQLLESGGGLVQPGGSLRLSCAASGF QTVVTQEPSLSVSPGGTVTLTCGSS mAb405TFDTYAMNWVRQAPGKGLEWVARIR TGAVTTSNYANWVQQTPGQAPRGSKYNNYATYYADSVKDRFTISRDDSKS LIGGTDKRAPGVPDRFSGSLLGDKATLYLQMESLRAEDTAVYYCVRHGSFG ALTITGAQAEDEADYYCALWYSNHNHIVSWFAHWGQGTLVTVSS (SEQ ID WVFGGGTKLTVL (SEQ ID NO: 460) NO: 459)LCDR1: GSSTGAVTTSNYAN (SEQ HCDR1: FTFDTYAMN (SEQ ID NO: 461) ID NO: 464)HCDR2: RIRSKYNNYATYYADSVKD LCDR2: GTDKRAP (SEQ ID NO: 465)(SEQ ID NO: 462) LCDR3: ALWYSNHWV (SEQ ID NO:HCDR3: VRHGSFGNHIVSWFAH (SEQ 466) ID NO: 463) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-1FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTRKNYLAWYQQKPGQPDLENGNTIYDAKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDGYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: RRTFGGGTKVEIK (SEQ ID NO: 468) 467) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-2FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNNRTRKNYLAWYQQKPGQPDLENANTIYDAKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDVYGRY TDFTLTISSLQAEDVAVYYCKQSYSLYDVWGQGTLVTVSS (SEQ ID NO: RRTFGGGTKVEIK (SEQ ID NO: 470) 469) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-3FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTRKNYLAWYQQKPGQPDLENGNTIYDPKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDAYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: RRTFGGGTKVEIK (SEQ ID NO: 472) 471) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-4FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNGRTRKNYLAWYQQKPGQPDLEEGNTIYDAKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGTGAYMELSSLRSEDTAVYYCARDAYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: RRTFGGGTKVEIK (SEQ ID NO: 473) 429) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVPLGERATINCKS mAb-5FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTRKNYLAWYQQKPGQPDLENANTIYDAKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDNYGGY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: RRTFGGGTKVEIK (SEQ ID NO: 475) 474) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-6FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTRKNYLAWYQQKPGQPDLENGNTIYDPKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDGYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: LRTFGGGTKVEIK (SEQ ID NO: 477) 476) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-7FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTRKNYLAWYQQKPGQPDLENGNTIYDPKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDGYGRY TDFTLTISSLQAEDVAVYYCKQSYNFFDVWGQGTLVTVSS (SEQ ID NO: LRTFGGGTKVEIK (SEQ ID NO: 479) 478) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-8FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTRKNYLAWYQQKPGQPDLENGNTIYDPKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCAREGYGRY TDFTLTISSLQAEDVAVYYCKQSYFFYDVWGQGTLVTVSS (SEQ ID NO: RRAFGGGTKVEIK (SEQ ID NO: 481) 480) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-9FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTRKNYLAWYQQKPGQPDLENGNTIYDPKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDGYGRY TDFTLTISSLQAEDVAVYYCKQSYNYYDVWGQGTLVTVSS (SEQ ID NO: LRTFGGGTKLEIK (SEQ ID NO: 483) 482) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-10FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLNSRTRKNYLAWYQQKPGQSDLENGNTIYQPKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFTGSGSGAYMELSSLRSEDTAVYYCARDGYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDVWGQGTLVTVSS (SEQ ID NO: LRTFGGGTKVEIK (SEQ ID NO: 485) 484) AdimabQVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS mAb-11FNIKDYYMHWVRQAPGQRLEWMGWI SQSLLESRTGKNYLAWYQQKPGQPDLENGNTIYDPKFQGRVTITRDTSAST PKLLIYWASTRESGVPDRFSGSGSGAYMELSSLRSEDTAVYYCARDGYGRY TDFTLTISSLQAEDVAVYYCKQSYSFYDYWGQGTLVTVSS (SEQ ID NO: LRTFGGGTKVEIK (SEQ ID NO: 487) 486) CD3DIKLQQSGAELARPGASVKMSCKTSG VDDIQLTQSPAIMSASPGEKVTMTC bindingYTFTRYTMHWVKQRPGQGLEWIGYIN RASSSVSYMNWYQQKSGTSPKRWI domain inPSRGYTNYNQKFKDKATLTTDKSSSTA YDTSKVASGVPYRFSGSGSGTSYSL blinatumoYMQLSSLTSEDSAVYYCARYYDDHYC TISSMEAEDAATYYCQQWSSNPLTF mabLDYWGQGTTLTVSSVE (SEQ ID NO: GAGTKLELK (SEQ ID NO: 489) 488) scFv:DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 490) NovimmuneQVQLVESGGGVVQPGRSLRLSCAASG EIVLTQSPATLSLSPGERATLSCRAS 28F11FKFSGYGMHWVRQAPGKGLEWVAVI QSVSSYLAWYQQKPGQAPRLLIYDWYDGSKKYYVDSVKGRFTISRDNSKN ASNRATGIPARFSGSGSGTDFTLTISTLYLQMNSLRAEDTAVYYCARQMGY SLEPEDFAVYYCQQRSNWPPLTFGWHFDLWGRGTLVTVSS (SEQ ID NO: GGTKVEIK (SEQ ID NO: 492) 491)LCDR1: RASQSVSSYLA (SEQ ID HCDR1: GYGMH (SEQ ID NO: 493) NO: 496)HCDR2: VIWYDGSKKYYVDSVKG LCDR2: DASNRAT (SEQ ID NO: 497)(SEQ ID NO: 494) LCDR3: QQRSNWPPLT (SEQ ID NO:HCDR3: QMGYWHFDL (SEQ ID NO: 498) 495) NovimmuneEVQLLESGGGLVQPGGSLRLSCAASGF DFMLTQPHSVSESPGKTVIISCWYQ 27H5TFSSFPMAWVRQAPGKGLEWVSTISTS QRPGRAPTTVIFGVPDRFSGSIDRSSGGRTYYRDSVKGRFTISRDNSKNTLYL NSASLTISGLQTEDEADYYCFGGGTQMNSLRAEDTAVYYCAKFRQYSGGFD KLTVLGQPKAAPSVTLFPPSSEELQYWGQGTLVTVSS (SEQ ID NO: 499) (SEQ ID NO: 500) Glaxo mAbEVQLLESGGGLVQPGGSLRLSCAASGF DIQLTQPNSVSTSLGSTVKLSCTLSSTFSSFPMAWVRQAPGKGLEWVSTISTS GNIENNYVHWYQLYEGRSPTTMIYGGRTYYRDSVKGRFTISRDNSKNTLYL DDDKRPDGVPDRFSGSIDRSSNSAFQMNSLRAEDTAVYYCAKFRQYSGGFD LTIHNVAIEDEAIYFCHSYVSSFNVFYWGQGTLVTVSS (SEQ ID NO: 501) GGGTKLTVLR (SEQ ID NO: 502) EurekaDVQLVQSGAEVKKPGASVKVSCKASG DIVLTQSPATLSLSPGERATLSCRAS mAbYTFTRYTMHWVRQAPGQGLEWIGYIN QSVSYMNWYQQKPGKAPKRWIYDPSRGYTNYADSVKGRFTITTDKSTSTA TSKVASGVPARFSGSGSGTDYSLTIYMELSSLRSEDTATYYCARYYDDHYC NSLEAEDAATYYCQQWSSNPLTFGLDYWGQGTPVTVSS (SEQ ID NO: 503) GGTKVEIK (SEQ ID NO: 504) scFv:DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK (SEQ ID NO: 505) MuromonabQVQLVQSGGGVVQPGRSLRLSCKASG DDIQMTQSPSSLSASVGDRVTITCSYTFTRYTMHWVRQAPGKGLEWIGYIN ASSSVSYMNWYQQTPGKAPKRWIYPSRGYTNYNQKVKDRFTISRDNSKNTA DTSKLASGVPSRFSGSGSGTDYTFTIFLQMDSLRPEDTGVYFCARYYDDHYC SSLQPEDIATYYCQQWSSNPFTFGQLDYWGQGTPVTVSS (SEQ ID NO: 506) GTKLQIT (SEQ ID NO: 507) MacroGenicsQVQLVQSGGGVVQPGRSLRLSCKASG DIQMTQSPSSLSASVGDRVTITCSAS mAbYTFTRYTMHWVRQAPGKGLEWIGYIN SSVSYMNWYQQTPGKAPKRWIYD humanizedPSRGYTNYNQKFKDRFTISTDKSKSTA TSKLASGVPSRFSGSGSGTDYTFTIS OKT3FLQMDSLRPEDTAVYYCARYYDDHYC SLQPEDIATYYCQQWS SNPFTFGQGLDYWGQGTPVTVSS (SEQ ID NO: 508) TKLQITR (SEQ ID NO: 509) RocheEVQLLESGGGLVQPGGSLRLSCAASGF QAVVTQEPSLTVSPGGTVTLTCGSS CH2527TFSTYAMNWVRQAPGKGLEWVSRIRS TGAVTTSNYANWVQEKPGQAFRGKYNNYATYYADSVKGRFTISRDDSKN LIGGTNKRAPGTPARFSGSLLGGKATLYLQMNSLRAEDTAVYYCVRHGNFG ALTLSGAQPEDEAEYYCALWYSNLNSYVSWFAYWGQGTLVTVSS (SEQ ID WVFGGGTKLTVL (SEQ ID NO: 601) NO: 600)Regeneron EVQLVESGGGLVQPGRSLRLSCAASGF AEIVMTQSPATLSVSPGERATLSCRmAb (anti- TFDDYTMHWVRQAPGKGLEWVSGIS ASQSVSSNLAWYQQKPGQAPRLLI CD3/anti-WNSGSIGYADSVKGRFTISRDNAKKSL YGASTRATGIPARFSGSGSGTEFTLT CD20)YLQMNSLRAEDTALYYCAKDNSGYG ISSLQSEDFAVYYCQHYINWPLTFGHYYYGMDVWGQGTTVTVAS (SEQ ID GGTKVEIK (SEQ ID NO: 603) NO: 602) WuXiQVQLVQSGAEVKKPGSSVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS WBP3311_YSFTTYYIHWVRQAPGQGLEWMGWIF SQSLLNSRTRKNYLAWYQQKPGQP 2.166.48-z1PGNDNIKYSEKFKGRVTITADKSTSTA PKLLIYWASTRKSGVPDRFSGSGSGYMELSSLRSEDTAVYYCAIDSVSIYYF TDFTLTISSLQAEDVAVYYCTQSFILDYWGQGTLVTVSS (SEQ ID NO: 604) RTFGGGTKVEIK (SEQ ID NO: 605)HCDR1: GYSFTTYYIH (SEQ ID NO: LCDR1: KSSQSLLNSRTRKNYLA 606)(SEQ ID NO: 609) HCDR2: WIFPGNDNIKYSEKFKG (SEQLCDR2: WASTRKS (SEQ ID NO: 610) ID NO: 607)LCDR3: TQSFILRT (SEQ ID NO: 611) HCDR3: DSVSIYYFDY (SEQ ID NO: 608) WuXiQVQLVQSGAEVKKPGSSVKVSCKASG DIVMTQSPDSLAVSLGERATINCKS WBP3311_FAFTDYYIHWVRQAPGQGLEWMGWIS SQSLLNSRTRKNYLAWYQQKPGQP 2.306.4-z1PGNVNTKYNENFKGRVTITADKSTSTA PKLLIYWASTRQSGVPDRFSGSGSGYMELSSLRSEDTAVYYCARDGYSLYY TDFTLTISSLQAEDVAVYYCTQSHTFDYWGQGTLVTVSS (SEQ ID NO: 612) LRTFGGGTKVEIK (SEQ ID NO: 613)HCDR1: GFAFTDYYIH (SEQ ID NO: LCDR1: KSSQSLLNSRTRKNYLA 614)(SEQ ID NO: 617) HCDR2: WISPGNVNTKYNENFKG (SEQLCDR2: WASTRQS (SEQ ID NO: 618) ID NO: 615) LCDR3: TQSHTLRT (SEQ ID NO:HCDR3: DGYSLYYFDY (SEQ ID NO: 619) 616) ADLQ MAESGGGSVQTGGSLRLSCAYTASSVN/A mAb-1 CMAWFRQAPGKEREGVAVTREGLTKT GYADSVKGRFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELL ASTYNYWGQGTQVTV (SEQ ID NO: 620) ADLQMAESGGGSVQTGGSLRLSCAYTASSV N/A mAb-2 CMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGRFAISQDYAKKTLYLQMS SLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 621) ADLQ MAESGGGSVQTGGSLRLSCAYTASSV N/AmAb-3 CMAWFRQAPGKEREGVAVTREGLTQT GYADSVKGRFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELL ASTYNYWGQGTQVTV (SEQ ID NO: 622) ADLQMAESGGGSVQTGGSLRLSCAYTASSV N/A mAb-4 CMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGRFAISQDYAKKTLYLQMS SLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 623) Where the VL and LCDR sequences arenoted as “N/A,” the antigen-binding site is an sdAb having a VH (e.g.,VHH) only.

In certain embodiments, the second antigen-binding site comprises a VHthat comprises an amino acid sequence at least 60% (e.g., at least 70%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) identical to the VH of an antibodydisclosed in Table 3, and a VL that comprises an amino acid sequence atleast 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto the VL of the same antibody disclosed in Table 3. In certainembodiments, the antigen-binding site comprises the HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3, determined under Kabat (see Kabat et al.,(1991) Sequences of Proteins of Immunological Interest, NIH PublicationNo. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum R M et al.,(1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) TheImmunologist, 7, 132-136), or any other CDR determination method knownin the art, of the VH and/or VL sequences of an antibody disclosed inTable 3. In certain embodiments, the antigen-binding site comprises theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibodydisclosed in Table 3. In certain embodiments, the antigen-binding sitecomprises the VH and VL sequences of an antibody disclosed in Table 3.

In certain embodiments, the second antigen-binding site that binds CD3is derived from CNG-CD3-1. In certain embodiments, the secondantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 414, 416, and 417, respectively, anda VL comprising LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ IDNOs: 419, 420, and 421, respectively. In certain embodiments, the secondantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 415, 416, and 418, respectively, anda VL comprising LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ IDNOs: 419, 420, and 421, respectively. In certain embodiments, the secondantigen-binding site comprises a VH comprising an amino acid sequence atleast 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto SEQ ID NO: 412, and a VL that comprising an amino acid sequence atleast 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto SEQ ID NO: 413. In certain embodiments, the VH and the VL of thesecond antigen-binding site comprise the amino acid sequences of SEQ IDNOs: 412 and 413, respectively.

In certain embodiments, the second antigen-binding site that binds CD3is derived from CNG-CD3-2. In certain embodiments, the secondantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 414, 416, and 425, respectively, anda VL comprising LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ IDNOs: 419, 420, and 421, respectively. In certain embodiments, the secondantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 415, 416, and 426, respectively, anda VL comprising LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ IDNOs: 419, 420, and 421, respectively. In certain embodiments, the secondantigen-binding site comprises a VH comprising an amino acid sequence atleast 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto SEQ ID NO: 424, and a VL that comprising an amino acid sequence atleast 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto SEQ ID NO: 413. In certain embodiments, the VH and the VL of thesecond antigen-binding site comprise the amino acid sequences of SEQ IDNOs: 424 and 413, respectively.

In certain embodiments, the second antigen-binding site that binds CD3is derived from CNG-CD3-3. In certain embodiments, the secondantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 414, 431, and 417, respectively, anda VL comprising LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ IDNOs: 419, 420, and 432, respectively. In certain embodiments, the secondantigen-binding site comprises a VH comprising HCDR1, HCDR2, and HCDR3sequences set forth in SEQ ID NOs: 415, 431, and 418, respectively, anda VL comprising LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ IDNOs: 419, 420, and 432, respectively. In certain embodiments, the secondantigen-binding site comprises a VH comprising an amino acid sequence atleast 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto SEQ ID NO: 429, and a VL that comprising an amino acid sequence atleast 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto SEQ ID NO: 430. In certain embodiments, the VH and the VL of thesecond antigen-binding site comprise the amino acid sequences of SEQ IDNOs: 429 and 430, respectively.

Such antigen-binding site may take the form of scFv. In certainembodiments, the VH is positioned C-terminal to the VL. In certainembodiments, the VH is positioned N-terminal to the VL. In certainembodiments, the VH and the VL are linked by a peptide linker, forexample, a linker disclosed in subsection D below titled “Linkers.” Incertain embodiments, the second antigen-binding site comprises the aminoacid sequence of SEQ ID NO: 422, 427, or 433. To stabilize the scFv, theamino acid residues at position 44 of the VH and at position 100 of theVL (under Kabat numbering) can be substituted by Cys, therebyfacilitating the formation of a disulfide bond between the VH and theVL. Accordingly, in certain embodiments, the VH and VL comprise Cys atpositions 100 and 44, respectively. In certain embodiments, the secondantigen-binding site comprises the amino acid sequence of SEQ ID NO:423, 428, or 434.

In other embodiments, the second antigen-binding site comprises an sdAbcomprising a VH comprising complementarity determining regions HCDR1,HCDR2, and HCDR3. In certain embodiments, the VH comprises an amino acidsequence at least 60% (e.g., at least 70%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the VH of an sdAb antibody provided in Table 3. In certainembodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences ofthe antibody provided in Table 3. In certain embodiments, the VHcomprises the amino acid sequence of the VH of an sdAb provided in Table3.

In certain embodiments, the second antigen-binding site competes forbinding CD3 (e.g., human CD3 and/or Macaca CD3) with an antibody orantigen-binding fragment thereof comprising the VH, VL and/or scFvsequences provided in Table 3.

In certain embodiments, the second antigen-binding site of themulti-specific binding protein binds CD3 (e.g., human CD3 and/or MacacaCD3) with a dissociation constant (K_(D)) of about 0.1 nM-about 1 μM.The K_(D) can be measured by a method known in the art. In certainembodiments, the K_(D) is measured by SPR to CD3 or an extracellularfragment thereof immobilized on a chip. In certain embodiments, theK_(D) is measured by flow cytometry to CD3 expressed on the surface ofcells, for example, following the method described in Example 6 below.

In certain embodiments, the second antigen-binding site binds CD3 with aK_(D) lower than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM,40 pM, 30 pM, 20 pM, or 10 pM. For example, in certain embodiments, thefirst antigen-binding site binds CD3 with a K_(D) about 10 pM-about 1nM, about 10 pM-about 0.9 nM, about 10 pM-about 0.8 nM, about 10pM-about 0.7 nM, about 10 pM-about 0.6 nM, about 10 pM nM-about 0.5 nM,about 10 pM-about 0.4 nM, about 10 pM-about 0.3 nM, about 10 pM-about0.2 nM, about 10 pM-about 0.1 nM, about 10 pM-about 50 pM, 0.1 nM-about10 nM, about 0.1 nM-about 9 nM, about 0.1 nM-about 8 nM, about 0.1nM-about 7 nM, about 0.1 nM-about 6 nM, about 0.1 nM-about 5 nM, about0.1 nM-about 4 nM, about 0.1 nM-about 3 nM, about 0.1 nM-about 2 nM,about 0.1 nM-about 1 nM, about 0.1 nM-about 0.5 nM, about 0.5 nM-about10 nM, about 1 nM-about 10 nM, about 2 nM-about 10 nM, about 3 nM-about10 nM, about 4 nM-about 10 nM, about 5 nM-about 10 nM, about 6 nM-about10 nM, about 7 nM-about 10 nM, about 8 nM-about 10 nM, or about 9nM-about 10 nM.

It is understood that a greater K_(D) (i.e., a lower affinity to CD3)may be desirable in the context of a multi-specific binding protein.Without wishing to be bound by theory, it is contemplated that amulti-specific binding protein having a very high affinity to CD3 mayresult in excessive cytokine release which narrows the therapeuticwindow. Accordingly, in certain embodiments, the second antigen-bindingsite binds CD3 (e.g., human CD3, e.g., human CD3ε) with a K_(D) greaterthan or equal to 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM,10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100nM. In certain embodiments, the second antigen-binding site binds CD3with a K_(D) about 1 nM-about 100 nM, about 1 nM-about 90 nM, about 1nM-about 80 nM, about 1 nM-about 70 nM, about 1 nM-about 60 nM, about 1nM-about 50 nM, about 1 nM-about 40 nM, about 1 nM-about 30 nM, about 1nM-about 20 nM, about 1 nM-about 10 nM, about 10 nM-about 100 nM, about10 nM-about 90 nM, about 10 nM-about 80 nM, about 10 nM-about 70 nM,about 10 nM-about 60 nM, about 10 nM-about 50 nM, about 10 nM-about 40nM, about 10 nM-about 30 nM, or about 10 nM-about 20 nM.

It is understood that the binding affinity to CD3 of the secondantigen-binding site alone may be different from the binding affinity ofthe same antigen-binding site in the context of the multi-specificbinding protein disclosed herein, possibly due to the conformationalrestraint from the other domains. The context-dependent binding affinityis described in the subsection E below titled “Binding Affinity”

In certain embodiments, the second antigen-binding site, when present inthe form of an Fab, has a melting temperature of at least 60° C., atleast 65° C., at least 70° C., at least 75° C., or at least 80° C. Incertain embodiments, the second antigen-binding site, when present inthe form of an Fab, has a melting temperature in the range of 60-85° C.,60-80° C., 60-75° C., 60-70° C., 60-65° C., 65-85° C., 65-80° C., 65-75°C., 65-70° C., 70-85° C., 70-80° C., 70-75° C., 75-85° C., 75-80° C., or80-85° C.

C. Construct Formats

The first, second, and third antigen-binding sites may take variousforms. In certain embodiments, the first, second, and/or thirdantigen-binding sites comprises two antibody variable domains (e.g., aVH and a VL). The VH and the VL can be mutated to introduce a disulfidebond (e.g., between H44 and L100) that stabilizes the antigen-bindingsite (see, Zhao et al. (2010) Int. J. Mol. Sci., 12(1):1-11). In certainembodiments, the first, second, and/or third antigen-binding sitescomprises a single antibody variable domain (e.g., an sdAb).

In an antigen-binding site that contains a VH and a VL, the VH and theVL can be linked to form an scFv. The VH can be positioned N-terminal orC-terminal to the VL. The VH and the VL are typically linked through alinker, such as a peptide linker. Exemplary sequences of peptide linkersare provided in subsection D below titled “Linkers.” In certainembodiments, the VH of an antigen-binding domain is connected to the VLof the antigen-binding domain through a peptide linker having an aminoacid sequence listed in Table 4. In particular embodiments, the VH of anantigen-binding domain is connected to the VL of the antigen-bindingdomain through a peptide linker having the amino acid sequence of SEQ IDNO: 298, 299, or 302, wherein the VH is positioned N-terminal to the VL.In other particular embodiments, the VH of an antigen-binding domain isconnected to the VL of the antigen-binding domain through a peptidelinker having the amino acid sequence of SEQ ID NO: 298, 299, or 302,wherein the VH is positioned C-terminal to the VL.

Alternatively, the VH and the VL may be present on separate polypeptidechains, and the formation of a VH-VL complex may be facilitated byadditional domains, such as antibody constant regions CH1 and CL.Accordingly, in certain embodiments, the multi-specific binding proteincomprises an Fab comprising a VH and a VL disclosed herein.

In certain embodiments, a multi-specific binding protein of the presentinvention comprises a first antigen-binding site comprising a singleantibody variable domain, a second antigen-binding site comprising asingle antibody variable domain, and a third antigen-binding sitecomprising a single antibody variable domain. In certain embodiments,the multi-specific binding protein comprises a first antigen-bindingsite in an sdAb format, a second antigen-binding site in an sdAb format,and a third antigen-binding site in an sdAb format.

In certain embodiments, a multi-specific binding protein of the presentinvention comprises a first antigen-binding site comprising a singleantibody variable domain, a second antigen-binding site comprising asingle antibody variable domain, and a third antigen-binding sitecomprising two antibody variable domains. In certain embodiments, themulti-specific binding protein comprises a first antigen-binding site inan sdAb format, a second antigen-binding site in an sdAb format, and athird antigen-binding site in an scFv format.

In certain embodiments, a multi-specific binding protein of the presentinvention comprises a first antigen-binding site comprising a singleantibody variable domain, a second antigen-binding site comprising twoantibody variable domains, and a third antigen-binding site comprising asingle antibody variable domain. In certain embodiments, themulti-specific binding protein comprises a first antigen-binding site inan sdAb format, a second antigen-binding site in an scFv format, and athird antigen-binding site in an sdAb format.

In certain embodiments, a multi-specific binding protein of the presentinvention comprises a first antigen-binding site comprising a singleantibody variable domain, a second antigen-binding site comprising twoantibody variable domains, and a third antigen-binding site comprisingtwo antibody variable domains. In certain embodiments, themulti-specific binding protein comprises a first antigen-binding site inan sdAb format, a second antigen-binding site in an scFv format, and athird antigen-binding site in an scFv format.

In certain embodiments, a multi-specific binding protein of the presentinvention comprises a first antigen-binding site comprising two antibodyvariable domains, a second antigen-binding site comprising a singleantibody variable domain, and a third antigen-binding site comprising asingle antibody variable domain. In certain embodiments, themulti-specific binding protein comprises a first antigen-binding site inan scFv format, a second antigen-binding site in an sdAb format, and athird antigen-binding site in an sdAb format.

In certain embodiments, a multi-specific binding protein of the presentinvention comprises a first antigen-binding site comprising two antibodyvariable domains, a second antigen-binding site comprising a singleantibody variable domain, and a third antigen-binding site comprisingtwo antibody variable domains. In certain embodiments, themulti-specific binding protein comprises a first antigen-binding site inan scFv format, a second antigen-binding site in an sdAb format, and athird antigen-binding site in an scFv format.

In certain embodiments, a multi-specific binding protein of the presentinvention comprises a first antigen-binding site comprising two antibodyvariable domains, a second antigen-binding site comprising two antibodyvariable domains, and a third antigen-binding site comprising a singleantibody variable domain. In certain embodiments, the multi-specificbinding protein comprises a first antigen-binding site in an scFvformat, a second antigen-binding site in an scFv format, and a thirdantigen-binding site in an sdAb format.

In certain embodiments, a multi-specific binding protein of the presentinvention comprises a first antigen-binding site comprising two antibodyvariable domains, a second antigen-binding site comprising two antibodyvariable domains, and a third antigen-binding site comprising twoantibody variable domains. In certain embodiments, the multi-specificbinding protein comprises a first antigen-binding site in an scFvformat, a second antigen-binding site in an scFv format, and a thirdantigen-binding site in an scFv format.

The three antigen-binding sites of the multi-specific binding proteincan be linked in any one of the following orientations in anamino-to-carboxyl direction:

(i) the first antigen-binding site (e.g., CD19 binding domain)—thesecond antigen-binding site (e.g., CD3 binding domain)—the thirdantigen-binding site (serum albumin binding domain);(ii) the first antigen-binding site (e.g., CD19 binding domain)—thethird antigen-binding site (serum albumin binding domain)—the secondantigen-binding site (e.g., CD3 binding domain);(iii) the second antigen-binding site (e.g., CD3 binding domain)—thefirst antigen-binding site (e.g., CD19 binding domain)—the thirdantigen-binding site (serum albumin binding domain);(iv) the second antigen-binding site (e.g., CD3 binding domain)—thethird antigen-binding site (serum albumin binding domain)—the firstantigen-binding site (e.g., CD19 binding domain);(v) the third antigen-binding site (serum albumin binding domain)—thefirst antigen-binding site (e.g., CD19 binding domain)—the secondantigen-binding site (e.g., CD3 binding domain); and(vi) the third antigen-binding site (serum albumin binding domain)—thesecond antigen-binding site (e.g., CD3 binding domain)—the firstantigen-binding site (e.g., CD19 binding domain), wherein the dashesabove represent a peptide bond and/or a linker (e.g., peptide linker).

In certain embodiments, the third antigen-binding site is not positionedbetween the first antigen-binding site and the second antigen-bindingsite. It is contemplated that constructs having such formats havefavorable therapeutic efficacy and in vivo half-life. In certainembodiments, the third antigen-binding site is positioned N-terminal toboth the first antigen-binding site and the second antigen-binding siteor C-terminal to both the first antigen-binding site and the secondantigen-binding site. In certain embodiments, the third antigen-bindingsite is positioned N-terminal to both the first antigen-binding site andthe second antigen-binding site. In certain embodiments, the thirdantigen-binding site is positioned C-terminal to both the firstantigen-binding site and the second antigen-binding site.

The position (N-terminal or C-terminal) of one antigen-binding siterelative to another is determined under the definitions of “N-terminal”and “C-terminal” as known in the art if a single polypeptide chaincomprises both antigen-binding sites. It is understood that if anantigen-binding site comprises two separate polypeptide chains, itsposition (N-terminal or C-terminal) relative to another antigen-bindingsite (either having a single polypeptide chain or two polypeptidechains) can be similarly determined if a single polypeptide chaincomprises at least one polypeptide chain of the former and at least onepolypeptide chain of the latter. It is further understood that ifantigen-binding site A is N-terminal to antigen-binding site B andantigen-binding site B is N-terminal to antigen-binding site C, it isdeemed that antigen-binding site A is positioned N-terminal toantigen-binding site C even if antigen-binding sites A and C are notpresent in any single, common polypeptide chain. More complex structuresof multi-specific binding proteins are also contemplated, some of whichmay have orientations difficult to characterize using the terms of“N-terminal” and “C-terminal” as described above due to, for example,different relative positions of two antigen-binding sites on onepolypeptide chain versus another polypeptide chain, or the presence of aloop structure.

According to the present invention, the multi-specific binding proteinsand their constituent binding domains are in the form of one or morepolypeptides. Such polypeptides may include proteinaceous parts andnon-proteinaceous parts (e.g., chemical linkers or chemicalcross-linking agents such as glutaraldehyde). In certain embodiments, amulti-specific binding protein of the present invention includes a firstantigen-binding site, a second antigen-binding site, and a thirdantigen-binding site, all of which are linked together to form a singlepolypeptide chain. In certain embodiments, the first, second, and thirdantigen-binding sites take the forms of scFv and/or sdAb, for example,in a combination as described above, to form a single polypeptide chain.

D. Linkers

As noted above, the antigen-binding sites of the multi-specific bindingproteins of the present invention can be linked through a peptide bondor a linker (e.g., peptide linker). In certain embodiments, at least twoadjacent antigen-binding sites are connected by a linker (e.g., peptidelinker). In certain embodiments, each two adjacent antigen-binding sitesare connected by a linker (e.g., peptide linker).

In certain embodiments, the three antigen-binding sites of themulti-specific binding protein can be linked by linkers (e.g., peptidelinkers) denoted as L₁ and L₂ in any one of the following orientationsin an amino-to-carboxyl direction:

(i) the first antigen-binding site (e.g., CD19 binding domain)—L₁— thesecond antigen-binding site (e.g., CD3 binding domain)—L₂— the thirdantigen-binding site (serum albumin binding domain);(ii) the first antigen-binding site (e.g., CD19 binding domain)—L₁— thethird antigen-binding site (serum albumin binding domain)—L₂— the secondantigen-binding site (e.g., CD3 binding domain);(iii) the second antigen-binding site (e.g., CD3 binding domain)—L₁— thefirst antigen-binding site (e.g., CD19 binding domain)—L₂— the thirdantigen-binding site (serum albumin binding domain);(iv) the second antigen-binding site (e.g., CD3 binding domain)—L₁— thethird antigen-binding site (serum albumin binding domain)—L₂— the firstantigen-binding site (e.g., CD19 binding domain);(v) the third antigen-binding site (serum albumin binding domain)—L₁—the first antigen-binding site (e.g., CD19 binding domain)—L₂— thesecond antigen-binding site (e.g., CD3 binding domain); and(vi) the third antigen-binding site (serum albumin binding domain)—L₁—the second antigen-binding site (e.g., CD3 binding domain)—L₂— the firstantigen-binding site (e.g., CD19 binding domain).

It is appreciated that in a given construct, L₁, L₂, or both L₁ and L₂may be replaced with a peptide bond.

It is understood that if a single polypeptide chain comprises twoadjacent antigen-binding sites, the peptide linker connecting the twoantigen-binding sites represents the amino acid sequence between them.If an antigen-binding site comprises two separate polypeptide chains,one of which is present in a single, common polypeptide as an adjacentantigen-binding site or a polypeptide chain thereof, the peptide linkerconnecting the two antigen-binding sites represents the amino acidsequence between them in the common, single polypeptide.

In certain embodiments, the linkers L₁ and L₂ are peptide linkers.Suitable lengths of L₁ and L₂ can be independently selected. Forexample, in certain embodiments, L₁ and/or L₂ are about 50 or less aminoacid residues in length. In certain embodiments, L₁ consists of about 50or less amino acid residues. In certain embodiments, L₁ consists ofabout 20 or less amino acid residues. In certain embodiments, L₂consists of about 50 or less amino acid residues. In certainembodiments, L₂ consists of about 20 or less amino acid residues. Incertain embodiments, L₁ and L₂ independently consist of about 50 or lessamino acid residues. In certain embodiments, L₁ and L₂ independentlyconsist of about 20 or less amino acid residues.

In some embodiments, peptide linkers L₁ and L₂ have an optimized lengthand/or amino acid composition. In some embodiments, L₁ and L₂ are of thesame length and have the same amino acid composition. In otherembodiments, L₁ and L₂ are different. In certain embodiments, L₁ and/orL₂ are “short,” i.e., consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12amino acid residues. Thus, in certain instances, the linkers consist ofabout 12 or less amino acid residues. In certain embodiments, L₁ and/orL₂ are “long,” e.g., consist of 15, 20 or 25 amino acid residues. Insome embodiments, L₁ and/or L₂ consist of about 3 to about 15, forexample 8, 9 or 10 contiguous amino acid residues.

Regarding the amino acid composition of L₁ and L₂, peptides are selectedwith properties that confer flexibility to multi-specific bindingprotein of the present invention, do not interfere with the bindingdomains as well as resist cleavage from proteases. For example, glycineand serine residues generally provide protease resistance. Examples ofthe linkers suitable for linking the domains in the multi-specificbinding protein include but are not limited to (GS)_(n), (GGS)_(n),(GGGS)_(n), (GGSG)_(n), (GGSGG)_(n), and (GGGGS)_(n), wherein n is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Insome embodiments, L₁ and/or L₂ are independently selected from thepeptide sequences listed in Table 4. In some embodiments, L₁ and/or L₂are independently selected from SEQ ID NOs: 292, 293, 294, 295, 296,297, 298, 299, 300, 301, or 302. In some embodiments, L₁ and/or L₂ areindependently selected from SEQ ID NOs: 298, 299, and 302. In someembodiments, L₁ and/or L₂ comprise the amino acid sequence of SEQ ID NO:298, 299, or 302. In some embodiments, L₁ and/or L₂ consist of the aminoacid sequence of 298, 299, or 302. In some embodiments, L₁ and L₂ eachcomprise the amino acid sequence of SEQ ID NO: 298, 299, or 302. In someembodiments, L₁ and L₂ each consist of the amino acid sequence of SEQ IDNO: 298, 299, or 302.

TABLE 4 Sequences of Exemplary Peptide Linkers SEQ ID Linker NO LengthAmino Acid Sequence (GS)₁₀ 292  20 GSGSGSGSGSGSGSGSGSGS (GGS)₁₀ 293  30GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (GGGS)₁₀ 294  40GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS GGGS (GGSG)₁₀ 295  40GGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSG GGSG (GGSGG)₁₀ 296  50GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGG (GGGGS)₁₀ 297  50GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGS (GGGGS)₄ 298  20GGGGSGGGGSGGGGSGGGGS (GGGGS)₃ 299  15 GGGGSGGGGSGGGGS (GGGGS)₂₀ 300 100GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (GGSGG)₂₀ 301 100GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG Assymetrical 302   9 GGGGSGGGS linker

A linker, such as a peptide linker disclosed herein, can also be used toconnect the VH and VL of an scFv, as mentioned in subsection C abovetitled “Construct Formats.”

In certain embodiments, the multi-specific binding protein furthercomprises a tag peptide, for example, a Flag tag, a 6×His tag, or 10×Histag (HHHHHHHHHH, SEQ ID NO: 711). Such tag peptides are useful forpurifying the multi-specific binding protein. In certain embodiments,the tag peptide (e.g., the 10×His tag) is positioned at the C-terminusof the multi-specific binding protein. In certain embodiments, the tagpeptide (e.g., the 10×His tag) is positioned at the N-terminus of themulti-specific binding protein.

E. Binding Affinity

In certain embodiments, the multi-specific binding protein binds CD19(e.g., human CD19), CD3 (e.g., human CD3 and/or Macaca CD3), and/orserum albumin (e.g., HSA) with a K_(D) in the range of about 0.1nM-about 100 μM. The K_(D) can be measured by a method known in the art,such as by SPR or by flow cytometry as described in Example 1 or 6below.

In certain embodiments, the multi-specific binding protein binds CD19,CD3, and/or serum albumin with a K_(D) lower than or equal to (i.e.,binding stronger than or equal to) 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50pM, 40 pM, 30 pM, 20 pM, or 10 pM. For example, in certain embodiments,the multi-specific binding protein binds CD19, CD3, and/or serum albuminwith a K_(D) in the range of about 10 pM-about 1 nM, about 10 pM-about0.9 nM, about 10 pM-about 0.8 nM, about 10 pM-about 0.7 nM, about 10pM-about 0.6 nM, about 10 pM nM-about 0.5 nM, about 10 pM-about 0.4 nM,about 10 pM-about 0.3 nM, about 10 pM-about 0.2 nM, about 10 pM-about0.1 nM, about 10 pM-about 50 pM, 0.1 nM-about 10 nM, about 0.1 nM-about9 nM, about 0.1 nM-about 8 nM, about 0.1 nM-about 7 nM, about 0.1nM-about 6 nM, about 0.1 nM-about 5 nM, about 0.1 nM-about 4 nM, about0.1 nM-about 3 nM, about 0.1 nM-about 2 nM, about 0.1 nM-about 1 nM,about 0.1 nM-about 0.5 nM, about 0.5 nM-about 10 nM, about 1 nM-about 10nM, about 2 nM-about 10 nM, about 3 nM-about 10 nM, about 4 nM-about 10nM, about 5 nM-about 10 nM, about 6 nM-about 10 nM, about 7 nM-about 10nM, about 8 nM-about 10 nM, or about 9 nM-about 10 nM.

In certain embodiments, the multi-specific binding protein binds CD19,CD3, and/or serum albumin with a K_(D) greater than or equal to (i.e.,binding weaker than or equal to) 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60nM, 70 nM, 80 nM, 90 nM, or 100 nM. In certain embodiments, themulti-specific binding protein binds CD19, CD3, and/or serum albuminwith a K_(D) in the range of about 10 nM-about 1000 nM, about 10nM-about 900 nM, about 10 nM-about 800 nM, about 10 nM-about 700 nM,about 10 nM-about 600 nM, about 10 nM-about 500 nM, about 10 nM-about400 nM, about 10 nM-about 300 nM, about 10 nM-about 200 nM, about 10nM-about 100 nM, about 10 nM-about 50 nM, about 50 nM-about 1000 nM,about 100 nM-about 1000 nM, about 200 nM-about 1000 nM, about 300nM-about 1000 nM, about 400 nM-about 1000 nM, about 500 nM-about 1000nM, about 600 nM-about 1000 nM, about 700 nM-about 1000 nM, about 800nM-about 1000 nM, or about 900 nM-about 1000 nM.

In certain embodiments, the K_(D) of binding to CD19 or CD3 is measuredin the absence of serum albumin (e.g., HSA). In certain embodiments, theK_(D) of binding to CD19 or CD3 is measured in substantial absence ofserum albumin (e.g., HSA). In certain embodiments, the K_(D) of bindingto CD19 or CD3 is measured in the presence of serum albumin (e.g., HSA),for example, in the presence of about 10 mg/mL, 15 mg/mL, 20 mg/mL, 25mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or 50 mg/mL serum albumin(e.g., HSA).

In certain embodiments, the multi-specific binding protein of thepresent disclosure binds CD19, CD3, and/or serum albumin with a similarK_(D) value to that of the respective antigen-binding site alone or amonoclonal antibody having the same antigen-binding site. In certainembodiments, the K_(D) value of the multi-specific binding protein toCD19, CD3, and/or serum albumin is increased by no more than 1.5 fold, 2fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold,15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, or 50fold compared to that of the respective antigen-binding site alone or amonoclonal antibody having the same antigen-binding site.

In certain embodiments, the multi-specific binding protein of thepresent disclosure binds CD19 and/or CD3 with a similar K_(D) value inthe presence of serum albumin to that in the absence or substantialabsence of serum albumin. In certain embodiments, the K_(D) value of themulti-specific binding protein for binding CD19 and/or CD3 in thepresence of serum albumin is increased by no more than 1.5 fold, 2 fold,3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, or 50 foldcompared to that in the absence or substantial absence of serum albumin.

F. Therapeutic Activities

The multi-specific binding protein disclosed herein is designed tosimultaneously bind B cells and T cells. Recruitment of T cellsfacilitates lysis of the B cells involving cytolytic synapse formationand delivery of perforin and granzymes. The engaged T cells are capableof serial target cell lysis, and are not affected by immune escapemechanisms interfering with peptide antigen processing and presentation,or clonal T cell differentiation; see, for example, WO2007042261A2.Accordingly, binding of the multi-specific binding proteins to thetarget B cells destroys the target cells and/or impairs the progressionof B cell related diseases.

Cytotoxicity mediated by multi-specific binding proteins of theinvention can be measured in various ways in vitro. Effector cells canbe e.g., stimulated enriched (human) CD8 positive T cells orunstimulated (human) peripheral blood mononuclear cells (PBMC). If thetarget cells are of macaque origin or express or are transfected withmacaque target cell surface antigen which is bound by the first domain,the effector cells should also be of macaque origin such as a macaque Tcell line, e.g., 4119LnPx. The target cells should express the proteintargeted by the first antigen-binding site, e.g., CD19, HER2, BCMA,CD33, or EGFR. In some embodiments, the target cells should expressCD19, e.g., human or macaque CD19.

In some embodiments, the target cells can be a cell line (such as CHO)which is stably or transiently transfected with CD19. Alternatively, insome embodiments, the target cells can be a cell line naturallyexpressing CD19, such as B lymphocytes. The effector to target cell(E:T) ratio is usually about 10:1, but can also vary. Killing of thetarget cells can be measured in a ⁵¹Cr-release assay (incubation time ofabout 18 hours) or in a in a FACS-based cytotoxicity assay (incubationtime of about 48 hours). Other methods of measuring cell death arewell-known to the skilled person, such as MTT or MTS assays, ATP-basedassays including bioluminescent assays, the sulforhodamine B (SRB)assay, WST assay, clonogenic assay and the ECIS technology.

In certain embodiments, the cytotoxic activity mediated by themulti-specific binding protein disclosed herein is measured in acell-based cytotoxicity assay described above. It is represented by theEC₅₀ value, which corresponds to the half maximal effectiveconcentration (concentration of the multi-specific binding protein whichinduces a cytotoxic response halfway between the baseline and maximum).In certain embodiments, the EC₅₀ value of the multi-specific bindingproteins is ≤5000 pM, for example, ≤4000 pM, ≤3000 pM, ≤2000 pM, ≤1000pM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤20 pM, ≤10 pM,≤5 pM, ≤4 pM, ≤3 pM, ≤2 pM, or ≤1 pM.

It is understood that an EC₅₀ value is generally lower whenstimulated/enriched CD8⁺ T cells are used as effector cells, comparedwith unstimulated PBMC. It is further understood that the EC₅₀ value isgenerally lower when the target cells express a high level of the targetcell surface antigen compared with a low level of the target antigen.For example, when stimulated/enriched human CD8⁺ T cells are used aseffector cells (and either target cell surface antigen transfected cellssuch as CHO cells or target cell surface antigen positive human celllines are used as target cells), the EC₅₀ value of multi-specificbinding protein is ≤1000 pM, for example, ≤500 pM, ≤250 pM, ≤100 pM, ≤50pM, ≤10 pM, or ≤5 pM. When human PBMCs are used as effector cells, theEC₅₀ value of the multi-specific binding protein is ≤5000 pM, forexample, ≤4000 pM, ≤2000 pM, ≤1000 pM, ≤500 pM, ≤200 pM, ≤150 pM, 100pM, X50 pM, ≤10 pM, or ≤5 pM. When a macaque T cell line such asLnPx4119 is used as effector cells, and a macaque target cell surfaceantigen transfected cell line such as CHO cells is used as target cellline, the EC₅₀ value of the multi-specific binding protein is ≤2000 pM,for example, ≤1500 pM, ≤1000 pM, ≤500 pM, ≤300 pM, ≤250 pM, ≤100 pM, ≤50pM, ≤10 pM, or ≤5 pM.

Accordingly, in certain embodiments, the EC₅₀ value is measured usingstimulated/enriched human CD8⁺ T cells as effector cells. In certainembodiments, the EC₅₀ value is measured using human PBMCs as effectorcells. In certain embodiments, the EC₅₀ value is measured using amacaque T cell line such as LnPx4119 as effector cells and cells (e.g.,CHO cells) engineered to express macaque CD19 as target cells.

In certain embodiments, the multi-specific binding protein of thepresent invention does not induce or mediate lysis of cells that do notexpress CD19. The term “does not induce lysis” or “does not mediatelysis,” or grammatical equivalents thereof, means that themulti-specific binding protein, at a concentration of up to 500 nM, doesnot induce or mediate lysis of more than 30%, for example, no more than30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5% of cells that do notexpress CD19, whereby lysis of a cell line that expresses CD19 is set tobe 100%.

In certain embodiments, a multi-specific binding protein disclosedherein is more effective in killing CD19-expressing cells (e.g., cancercells) than the corresponding respective anti-CD19 or anti-CD3monoclonal antibody at the same molar concentration. In certainembodiments, the multi-specific binding protein is more effective inkilling CD19-expressing cells (e.g., cancer cells) than a combination ofthe corresponding respective anti-CD19 and anti-CD3 monoclonalantibodies each at the same molar concentration.

The cytotoxic activity of the multi-specific binding protein can bemeasured in the presence or absence of serum albumin (e.g., HSA). Incertain embodiments, the cytotoxic activity disclosed above is measuredin the absence of serum albumin (e.g., HSA). In certain embodiments, thecytotoxic activity disclosed above is measured in substantial absence ofserum albumin (e.g., HSA). In certain embodiments, the cytotoxicactivity disclosed above is measured in the presence of serum albumin(e.g., HSA), for example, in the presence of about 10 mg/mL, 15 mg/mL,20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or 50 mg/mLserum albumin (e.g., HSA).

In certain embodiments, the multi-specific binding protein of thepresent disclosure kills CD19-expressing cells with a similar EC₅₀ valuein the presence of serum albumin to that in the absence or substantialabsence of serum albumin. In certain embodiments, the EC₅₀ value of themulti-specific binding protein for killing CD19-expressing cells in thepresence of serum albumin is increased by no more than 1.5 fold, 2 fold,3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, or 50 foldcompared to that in the absence or substantial absence of serum albumin.It is understood that the presence of serum albumin (e.g., about 10mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45mg/mL, or 50 mg/mL serum albumin) may also alter the EC₅₀ value of amulti-specific binding protein nonspecifically. The nonspecific effectcan be assessed by comparing the EC₅₀ values of a control protein, whichdoes not contain a serum albumin binding domain, in the presence andabsence of serum albumin. In certain embodiments, the fold change isoffset by the nonspecific effect of serum albumin on a control protein,such as a bispecific protein that binds CD19 and CD3.

G. Construct Size

In certain embodiments, the molecular weight of the multi-specificbinding protein is from about 40 kD to about 100 kD. In certainembodiments, the molecular weight of the multi-specific binding proteinis at least 60 kD, at least 65 kD, at least 70 kD, at least 75 kD, atleast 80 kD, at least 85 kD, at least 90 kD, or at least 95 kD. It isunderstood that smaller size generally contributes to faster diffusionand tissue penetration, but size reduction may not be as critical forthe purpose of treating the indications with substantial presence oftarget cells (e.g., cancer cells) in the blood.

In certain embodiments, the molecular weight of the multi-specificbinding protein is from about 40 kD to about 90 kD, from about 40 kD toabout 80 kD, from about 40 kD to about 70 kD, from about 40 kD to about60 kD, from about 40 kD to about 50 kD, from about 50 kD to about 100kD, from about 50 kD to about 90 kD, from about 50 kD to about 80 kD,from about 50 kD to about 70 kD, from about 50 kD to about 60 kD, fromabout 60 kD to about 100 kD, from about 60 kD to about 90 kD, from about60 kD to about 80 kD, from about 60 kD to about 70 kD, from about 65 kDto about 100 kD, from about 65 kD to about 90 kD, from about 65 kD toabout 80 kD, from about 65 kD to about 70 kD, from about 70 kD to about100 kD, from about 70 kD to about 90 kD, from about 70 kD to about 80kD, from about 80 kD to about 100 kD, from about 80 kD to about 90 kD,or from about 90 kD to about 100 kD. In certain embodiments, themulti-specific binding protein is lower than 40 kD. In certainembodiments, the multi-specific binding protein is about 50 kD-about 90kD, about 50 kD-about 80 kD, about 50 kD-about 70 kD, about 50 kD-about60 kD, about 60 kD-about 90 kD, about 60 kD-about 80 kD, about 60kD-about 70 kD, about 65 kD-about 90 kD, about 65 kD-about 80 kD, about65 kD-about 70 kD, about 70 kD-about 90 kD, or about 70 kD-about 80 kD.

H. Serum Half-Life

Fusion proteins have been developed to increase the in vivo half-life ofa small protein, particularly an antibody fragment. For example, fusionwith a heterodimeric antibody Fc region, such as an Fc with one or moremutations that extend the in vivo half-life, is described in U.S. PatentApplication Publication Nos. US20140302037A1, US20140308285A1, and PCTPublication Nos. WO2014144722A2, WO2014151910A1 and WO2015048272A1. Analternative strategy is fusion with human serum albumin (HSA) or anHSA-binding peptide (see, e.g., PCT Publication Nos. WO2013128027A1 andWO2014140358A1). The neonatal Fc receptor (FcRn) appears to be involvedin prolonging the life-span of albumin in circulation (see, Chaudhury etal. (2003) J. Exp. Med., 3: 315-22). Albumin and IgG bindnoncooperatively to distinct sites of FcRn and form a tri-molecular (seeid.). Binding of human FcRn to HSA and to human IgG is pH dependent,stronger at acidic pH and weaker at neutral or physiological pH (seeid.). This observation suggests that proteins and protein complexescontaining albumin, similar to those containing IgG (particularly Fc),are protected from degradation through pH-sensitive interaction withFcRn (see id.). Using surface plasmon resonance (SPR) to measure thecapacity of individual HSA domains to bind immobilized soluble humanFcRn, it has been shown that FcRn and albumin interact via the D-IIIdomain of albumin in a pH-dependent manner, on a site distinct from theIgG binding site (see, Chaudhury et al. (2006) Biochemistry 45:4983-90and PCT Publication No. WO2008068280A1).

The present disclosure provides multi-specific binding proteins withextended half-life. In certain embodiments, the multi-specific bindingprotein has a serum half-life of at least 24, 36, 48, 60, 72, 84, or 96hours. In certain embodiments, the multi-specific binding protein has aserum half-life of at least about 50 hours. In certain embodiments, themulti-specific binding protein has a serum half-life of at least about100 hours. Methods of measuring serum half-life are known in the art,and exemplary methods are described in Example 5. In certainembodiments, the serum half-life is measured in a non-human primate. Incertain embodiments, the serum half-life is measured in human.

In certain embodiments, 50 hours after intravenous administration to asubject, the serum concentration of the multi-specific binding proteinis at least 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, or at least 90% of the serumconcentration of the multi-specific binding protein 1 hour after theadministration in said subject.

In certain embodiments, the multi-specific binding protein has a serumhalf-life that is at least 20% longer than a control multi-specificbinding protein, wherein the control multi-specific binding proteinincludes a first domain identical to the first antigen-binding site ofthe multi-specific binding protein, a second domain identical to thesecond antigen-binding site of the multi-specific binding protein, butnot a third domain identical or substantially identical to the thirdantigen-binding site of the multi-specific binding protein. In certainembodiments, the control multi-specific binding protein is identical tothe multi-specific binding protein but for the absence of the thirdantigen-binding site. In certain embodiments, the serum half-life of themulti-specific binding protein is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, or at least 90% longerthan the serum half-life of the control multi-specific binding protein.In certain embodiments, the serum half-life of the multi-specificbinding protein is longer than the serum half-life of the controlmulti-specific binding protein by at least 2 fold, at least 3 fold, atleast 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, or at least 10 fold.

III. Methods of Preparation

The antibodies and multi-specific binding proteins described above canbe made using recombinant DNA technology well known to a skilled personin the art. For example, one or more isolated polynucleotides encodingthe antibody or the multi-specific binding protein can be ligated toother appropriate nucleotide sequences, including, for example, constantregion coding sequences, and expression control sequences, to produceconventional gene expression constructs (i.e., expression vectors)encoding the desired antibodies or multi-specific binding proteins.Production of defined gene constructs is within routine skill in theart.

Nucleic acids encoding desired antibodies or multi-specific bindingproteins can be incorporated (ligated) into expression vectors, whichcan be introduced into host cells through conventional transfection ortransformation techniques. Exemplary host cells are E. coli cells,Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293)cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells(COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myelomacells that do not otherwise produce IgG protein. Transformed host cellscan be grown under conditions that permit the host cells to express thegenes that encode the antibodies or multi-specific binding proteins.

Specific expression and purification conditions will vary depending uponthe expression system employed. For example, if a gene is to beexpressed in E. coli, it is first cloned into an expression vector bypositioning the engineered gene downstream from a suitable bacterialpromoter, e.g., Trp or Tac, and a prokaryotic signal sequence. Theexpressed protein may be secreted. The expressed protein may accumulatein refractile or inclusion bodies, which can be harvested afterdisruption of the cells by French press or sonication. The refractilebodies then are solubilized, and the protein may be refolded and/orcleaved by methods known in the art.

If the engineered gene is to be expressed in eukaryotic host cells,e.g., CHO cells, it is first inserted into an expression vectorcontaining a suitable eukaryotic promoter, a secretion signal, a poly Asequence, and a stop codon. Optionally, the vector or gene construct maycontain enhancers and introns. In embodiments involving fusion proteinscomprising an antibody or portion thereof, the expression vectoroptionally contains sequences encoding all or part of a constant region,enabling an entire, or a part of, a heavy or light chain to beexpressed. The gene construct can be introduced into eukaryotic hostcells using conventional techniques.

The antibodies or multi-specific binding protein disclosed herein maycomprise a single polypeptide chain. In this instance, a host cell canbe transfected with a single vector expressing the polypeptide (e.g.,containing an expression control sequence operably linked to anucleotide sequence encoding the polypeptide). Alternatively, theantibodies or multi-specific binding proteins disclosed herein maycomprise two or more polypeptides. In this instance, a host cell can beco-transfected with more than one expression vector, for example, oneexpression vector expressing each polypeptide. A host cell can also betransfected with a single expression vector that expresses the two ormore polypeptides. For example, the coding sequences of the two or morepolypeptides can be operably linked to different expression controlsequences (e.g., promoter, enhancer, and/or internal ribosome entry site(IRES)). The coding sequences of the two or more polypeptides can alsobe separated by a ribosomal skipping sequence or self-cleaving sequence,such as a 2A peptide.

In certain embodiments, in order to express an antibody ormulti-specific binding protein, an N-terminal signal sequence isincluded in the protein construct. Exemplary N-terminal signal sequencesinclude signal sequences from interleukin-2, CD-5, IgG kappa lightchain, trypsinogen, serum albumin, and prolactin.

After transfection, single clones can be isolated for cell bankgeneration using methods known in the art, such as limited dilution,ELISA, FACS, microscopy, or Clonepix. Clones can be cultured underconditions suitable for bio-reactor scale-up and maintained expressionof the antibodies or multi-specific binding proteins.

The antibodies or multi-specific binding proteins can be isolated andpurified using methods known in the art including centrifugation, depthfiltration, cell lysis, homogenization, freeze-thawing, affinitypurification, gel filtration, ion exchange chromatography, hydrophobicinteraction exchange chromatography, and mixed-mode chromatography.

IV. Pharmaceutical Compositions

The present disclosure also features pharmaceutical compositions thatcontain a therapeutically effective amount of the antibodies ormulti-specific binding proteins described herein. The composition can beformulated for use in a variety of drug delivery systems. One or morephysiologically acceptable excipients or carriers can also be includedin the composition for proper formulation. Suitable formulations for usein the present disclosure are found in Remington's PharmaceuticalSciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985.For a brief review of methods for drug delivery, see, e.g., Langer(Science 249:1527-1533, 1990).

In certain embodiments, a pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants (see,Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company,1990).

In certain embodiments, a pharmaceutical composition may containnanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles(See Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

In certain embodiments, a pharmaceutical composition may contain asustained- or controlled-delivery formulation. Techniques forformulating sustained- or controlled-delivery means, such as liposomecarriers, bio-erodible microparticles or porous beads and depotinjections, are also known to those skilled in the art.Sustained-release preparations may include, e.g., porous polymericmicroparticles or semipermeable polymer matrices in the form of shapedarticles, e.g., films, or microcapsules. Sustained release matrices mayinclude polyesters, hydrogels, polylactides, copolymers of L-glutamicacid and gamma ethyl-L-glutamate, poly (2-hydroxyethyl-inethacrylate),ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyric acid. Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art.

Pharmaceutical compositions containing an antibody or a multi-specificbinding protein disclosed herein can be presented in a dosage unit formand can be prepared by any suitable method. A pharmaceutical compositionshould be formulated to be compatible with its intended route ofadministration. Examples of routes of administration are intravenous(IV), intradermal, inhalation, transdermal, topical, transmucosal,intrathecal and rectal administration. In certain embodiments, arecombinant human sialidase, a recombinant human sialidase fusionprotein, or an antibody conjugate disclosed herein is administered by IVinfusion. In certain embodiments, a recombinant human sialidase, arecombinant human sialidase fusion protein, or an antibody conjugatedisclosed herein is administered by intratumoral injection. Usefulformulations can be prepared by methods known in the pharmaceutical art.For example, see Remington's Pharmaceutical Sciences, 18th ed. (MackPublishing Company, 1990). Formulation components suitable forparenteral administration include a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerin,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfate; chelating agents such as EDTA; buffers such asacetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier should be stable under theconditions of manufacture and storage, and should be preserved againstmicroorganisms. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol), and suitablemixtures thereof.

An intravenous drug delivery formulation may be contained in a syringe,pen, or bag. In certain embodiments, the bag may be connected to achannel comprising a tube and/or a needle. In certain embodiments, theformulation may be a lyophilized formulation or a liquid formulation. Incertain embodiments, the formulation may freeze-dried (lyophilized) andcontained in about 12-60 vials. In certain embodiments, the formulationmay be freeze-dried and 45 mg of the freeze-dried formulation may becontained in one vial. In certain embodiments, the about 40 mg-about 100mg of freeze-dried formulation may be contained in one vial. In certainembodiments, freeze dried formulation from 12, 27, or 45 vials arecombined to obtained a therapeutic dose of the protein in theintravenous drug formulation. In certain embodiments, the formulationmay be a liquid formulation and stored as about 250 mg/vial to about1,000 mg/vial. In certain embodiments, the formulation may be a liquidformulation and stored as about 600 mg/vial. In certain embodiments, theformulation may be a liquid formulation and stored as about 250 mg/vial.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as-is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 7 and 8, such as 7 to 7.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of the above-mentioned agent or agents. Thecomposition in solid form can also be packaged in a container for aflexible quantity.

In certain embodiments, the present disclosure provides a formulationwith an extended shelf life including the protein of the presentdisclosure, in combination with mannitol, citric acid monohydrate,sodium citrate, disodium phosphate dihydrate, sodium dihydrogenphosphate dihydrate, sodium chloride, polysorbate 80, water, and sodiumhydroxide.

In certain embodiments, an aqueous formulation is prepared including theprotein of the present disclosure in a pH-buffered solution. The bufferof this invention may have a pH ranging from about 4 to about 8, e.g.,from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or may havea pH of about 5.0 to about 5.2. Ranges intermediate to the above recitedpH's are also intended to be part of this disclosure. For example,ranges of values using a combination of any of the above recited valuesas upper and/or lower limits are intended to be included. Examples ofbuffers that will control the pH within this range include acetate (e.g.sodium acetate), succinate (such as sodium succinate), gluconate,histidine, citrate and other organic acid buffers.

In certain embodiments, the formulation includes a buffer system whichcontains citrate and phosphate to maintain the pH in a range of about 4to about 8. In certain embodiments the pH range may be from about 4.5 toabout 6.0, or from about pH 4.8 to about 5.5, or in a pH range of about5.0 to about 5.2. In certain embodiments, the buffer system includescitric acid monohydrate, sodium citrate, disodium phosphate dihydrate,and/or sodium dihydrogen phosphate dihydrate. In certain embodiments,the buffer system includes about 1.3 mg/ml of citric acid (e.g., 1.305mg/ml), about 0.3 mg/ml of sodium citrate (e.g., 0.305 mg/ml), about 1.5mg/ml of disodium phosphate dihydrate (e.g., 1.53 mg/ml), about 0.9mg/ml of sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about6.2 mg/ml of sodium chloride (e.g., 6.165 mg/ml). In certainembodiments, the buffer system includes 1-1.5 mg/ml of citric acid, 0.25to 0.5 mg/ml of sodium citrate, 1.25 to 1.75 mg/ml of disodium phosphatedihydrate, 0.7 to 1.1 mg/ml of sodium dihydrogen phosphate dihydrate,and 6.0 to 6.4 mg/ml of sodium chloride. In certain embodiments, the pHof the formulation is adjusted with sodium hydroxide.

A polyol, which acts as a tonicifier and may stabilize the antibody ormulti-specific binding protein, may also be included in the formulation.The polyol is added to the formulation in an amount which may vary withrespect to the desired isotonicity of the formulation. In certainembodiments, the aqueous formulation may be isotonic. The amount ofpolyol added may also be altered with respect to the molecular weight ofthe polyol. For example, a lower amount of a monosaccharide (e.g.,mannitol) may be added, compared to a disaccharide (such as trehalose).In certain embodiments, the polyol which may be used in the formulationas a tonicity agent is mannitol. In certain embodiments, the mannitolconcentration may be about 5 to about 20 mg/ml. In certain embodiments,the concentration of mannitol may be about 7.5 to 15 mg/ml. In certainembodiments, the concentration of mannitol may be about 10-14 mg/ml. Incertain embodiments, the concentration of mannitol may be about 12mg/ml. In certain embodiments, the polyol sorbitol may be included inthe formulation.

A detergent or surfactant may also be added to the formulation.Exemplary detergents include nonionic detergents such as polysorbates(e.g., polysorbates 20, 80 etc.) or poloxamers (e.g., poloxamer 188).The amount of detergent added is such that it reduces aggregation of theformulated antibody and/or minimizes the formation of particulates inthe formulation and/or reduces adsorption. In certain embodiments, theformulation may include a surfactant which is a polysorbate. In certainembodiments, the formulation may contain the detergent polysorbate 80 orTween 80. Tween 80 is a term used to describe polyoxyethylene (20)sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio CantorVerlag Aulendorf, 4th edi., 1996). In certain embodiments, theformulation may contain between about 0.1 mg/mL and about 10 mg/mL ofpolysorbate 80, or between about 0.5 mg/mL and about 5 mg/mL. In certainembodiments, about 0.1% polysorbate 80 may be added in the formulation.

In embodiments, the protein product of the present disclosure isformulated as a liquid formulation. The liquid formulation may bepresented at a 10 mg/mL concentration in either a USP/Ph Eur type I 50Rvial closed with a rubber stopper and sealed with an aluminum crimp sealclosure. The stopper may be made of elastomer complying with USP and PhEur. In certain embodiments, the liquid formulation may be diluted with0.9% saline solution.

In certain embodiments, the liquid formulation of the disclosure may beprepared as a 10 mg/mL concentration solution in combination with asugar at stabilizing levels. In certain embodiments the liquidformulation may be prepared in an aqueous carrier. In certainembodiments, a stabilizer may be added in an amount no greater than thatwhich may result in a viscosity undesirable or unsuitable forintravenous administration. In certain embodiments, the sugar may bedisaccharides, e.g., sucrose. In certain embodiments, the liquidformulation may also include one or more of a buffering agent, asurfactant, and a preservative.

In certain embodiments, the pH of the liquid formulation may be set byaddition of a pharmaceutically acceptable acid and/or base. In certainembodiments, the pharmaceutically acceptable acid may be hydrochloricacid. In certain embodiments, the base may be sodium hydroxide.

The aqueous carrier of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation. Illustrativecarriers include sterile water for injection (SWFI), bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

A preservative may be optionally added to the formulations herein toreduce bacterial action. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation.

The antibody or multi-specific binding protein may be lyophilized toproduce a lyophilized formulation including the proteins and alyoprotectant. The lyoprotectant may be sugar, e.g., disaccharides. Incertain embodiments, the lyoprotectant may be sucrose or maltose. Thelyophilized formulation may also include one or more of a bufferingagent, a surfactant, a bulking agent, and/or a preservative.

The amount of sucrose or maltose useful for stabilization of thelyophilized drug product may be in a weight ratio of at least 1:2protein to sucrose or maltose. In certain embodiments, the protein tosucrose or maltose weight ratio may be of from 1:2 to 1:5. In certainembodiments, the pH of the formulation, prior to lyophilization, may beset by addition of a pharmaceutically acceptable acid and/or base. Incertain embodiments the pharmaceutically acceptable acid may behydrochloric acid. In certain embodiments, the pharmaceuticallyacceptable base may be sodium hydroxide. Before lyophilization, the pHof the solution containing the protein of the present disclosure may beadjusted between 6 to 8. In certain embodiments, the pH range for thelyophilized drug product may be from 7 to 8.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The specific dose can be a uniform dose for each patient, for example,50-5,000 mg of protein. Alternatively, a patient's dose can be tailoredto the approximate body weight or surface area of the patient. Otherfactors in determining the appropriate dosage can include the disease orcondition to be treated or prevented, the severity of the disease, theroute of administration, and the age, sex and medical condition of thepatient. Further refinement of the calculations necessary to determinethe appropriate dosage for treatment is routinely made by those skilledin the art, especially in light of the dosage information and assaysdisclosed herein. The dosage can also be determined through the use ofknown assays for determining dosages used in conjunction withappropriate dose-response data. An individual patient's dosage can beadjusted as the progress of the disease is monitored. Blood levels ofthe targetable construct or complex in a patient can be measured to seeif the dosage needs to be adjusted to reach or maintain an effectiveconcentration. Pharmacogenomics may be used to determine whichtargetable constructs and/or complexes, and dosages thereof, are mostlikely to be effective for a given individual (Schmitz et al., ClinicaChimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

In general, dosages based on body weight are from about 0.01 μg to about100 mg per kg of body weight, such as about 0.01 μg to about 100 mg/kgof body weight, about 0.01 μg to about 50 mg/kg of body weight, about0.01 μg to about 10 mg/kg of body weight, about 0.01 μg to about 1 mg/kgof body weight, about 0.01 μg to about 100 μg/kg of body weight, about0.01 μg to about 50 μg/kg of body weight, about 0.01 μg to about 10μg/kg of body weight, about 0.01 μg to about 1 μg/kg of body weight,about 0.01 μg to about 0.1 μg/kg of body weight, about 0.1 μg to about100 mg/kg of body weight, about 0.1 μg to about 50 mg/kg of body weight,about 0.1 μg to about 10 mg/kg of body weight, about 0.1 μg to about 1mg/kg of body weight, about 0.1 μg to about 100 μg/kg of body weight,about 0.1 μg to about 10 μg/kg of body weight, about 0.1 μg to about 1μg/kg of body weight, about 1 μg to about 100 mg/kg of body weight,about 1 μg to about 50 mg/kg of body weight, about 1 μg to about 10mg/kg of body weight, about 1 μg to about 1 mg/kg of body weight, about1 μg to about 100 μg/kg of body weight, about 1 μg to about 50 μg/kg ofbody weight, about 1 μg to about 10 μg/kg of body weight, about 10 μg toabout 100 mg/kg of body weight, about 10 μg to about 50 mg/kg of bodyweight, about 10 μg to about 10 mg/kg of body weight, about 10 μg toabout 1 mg/kg of body weight, about 10 μg to about 100 μg/kg of bodyweight, about 10 μg to about 50 μg/kg of body weight, about 50 μg toabout 100 mg/kg of body weight, about 50 μg to about 50 mg/kg of bodyweight, about 50 μg to about 10 mg/kg of body weight, about 50 μg toabout 1 mg/kg of body weight, about 50 μg to about 100 μg/kg of bodyweight, about 100 μg to about 100 mg/kg of body weight, about 100 μg toabout 50 mg/kg of body weight, about 100 μg to about 10 mg/kg of bodyweight, about 100 μg to about 1 mg/kg of body weight, about 1 mg toabout 100 mg/kg of body weight, about 1 mg to about 50 mg/kg of bodyweight, about 1 mg to about 10 mg/kg of body weight, about 10 mg toabout 100 mg/kg of body weight, about 10 mg to about 50 mg/kg of bodyweight, about 50 mg to about 100 mg/kg of body weight.

Doses may be given once or more times daily, weekly, monthly or yearly,or even once every 2 to 20 years. Persons of ordinary skill in the artcan easily estimate repetition rates for dosing based on measuredresidence times and concentrations of the targetable construct orcomplex in bodily fluids or tissues. Administration of the presentinvention could be intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary,by perfusion through a catheter or by direct intralesional injection.This may be administered once or more times daily, once or more timesweekly, once or more times monthly, and once or more times annually.

V. Therapeutic Applications

It is contemplated that the antibodies or multi-specific bindingproteins can be used either alone or in combination with othertherapeutic agents.

A. Indications

The present disclosure provides methods for the treatment oramelioration of a proliferative disease, a tumorous disease, aninflammatory disease, an immunological disorder, an autoimmune disease,an infectious disease, a viral disease, an allergic reaction, aparasitic reaction, a graft-versus-host disease or a host-versus-graftdisease in a subject in need thereof, the method comprisingadministration of a multi-specific binding protein or an antibodydisclosed herein. In certain embodiments, the disease is associated withexpression or overexpression of the target protein expressed on thetarget cell.

In certain embodiments, the cancer to be treated is non-Hodgkin'slymphoma, such as a B-cell lymphoma. In certain embodiments, thenon-Hodgkin's lymphoma is a B-cell lymphoma, such as a diffuse largeB-cell lymphoma, primary mediastinal B-cell lymphoma, follicularlymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginalzone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma,Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia,chronic lymphocytic leukemia, or primary central nervous systemlymphoma. In certain other embodiments, the cancer to be treated ismultiple myeloma. In certain other embodiments, the cancer to be treatedis acute lymphoblastic leukemia (ALL). In certain embodiments, the ALLis relapsed/refractory adult and pediatric ALL.

B. Combination Therapies

The methods and compositions described herein can be used alone or incombination with other therapeutic agents and/or modalities. The termadministered “in combination,” as used herein, is understood to meanthat two (or more) different treatments are delivered to the subjectduring the course of the subject's affliction with the disorder, suchthat the effects of the treatments on the patient overlap at a point intime. In certain embodiments, the delivery of one treatment is stilloccurring when the delivery of the second begins, so that there isoverlap in terms of administration. This is sometimes referred to hereinas “simultaneous” or “concurrent delivery.” In other embodiments, thedelivery of one treatment ends before the delivery of the othertreatment begins. In certain embodiments of either case, the treatmentis more effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In certainembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

In one aspect, the present disclosure provides a method of treating asubject by the administration of a second therapeutic agent incombination with one or more of the multi-specific binding proteinsand/or antibodies that bind CD19 disclosed herein.

Exemplary therapeutic agents that may be used as part of a combinationtherapy in treating cancer, include, for example, radiation, mitomycin,tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine,mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin,nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed,daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane,nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone,aminoglutethimide, amsacrine, proglumide, elliptinium acetate,ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin,nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane,sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine,picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride,oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol,formestane, interferon-alpha, interferon-2 alpha, interferon-beta,interferon-gamma, colony stimulating factor-1, colony stimulatingfactor-2, denileukin diftitox, interleukin-2, luteinizing hormonereleasing factor and variations of the aforementioned agents that mayexhibit differential binding to its cognate receptor, and increased ordecreased serum half-life.

An additional class of agents that may be used as part of a combinationtherapy in treating cancer is immune checkpoint inhibitors. Thecheckpoint inhibitor may, for example, be selected from a PD-1antagonist, PD-L1 antagonist, CTLA-4 antagonist, adenosine A2A receptorantagonist, B7-H3 antagonist, B7-H4 antagonist, BTLA antagonist, KIRantagonist, LAG3 antagonist, TIM-3 antagonist, VISTA antagonist or TIGITantagonist.

In certain embodiments, the checkpoint inhibitor is a PD-1 or PD-L1inhibitor. PD-1 is a receptor present on the surface of T-cells thatserves as an immune system checkpoint that inhibits or otherwisemodulates T-cell activity at the appropriate time to prevent anoveractive immune response. Cancer cells, however, can take advantage ofthis checkpoint by expressing ligands, for example, PD-L1, that interactwith PD-1 on the surface of T-cells to shut down or modulate T-cellactivity. Exemplary PD-1/PD-L1 based immune checkpoint inhibitorsinclude antibody based therapeutics. Exemplary treatment methods thatemploy PD-1/PD-L1 based immune checkpoint inhibition are described inU.S. Pat. Nos. 8,728,474 and 9,073,994, and EP Patent No. 1537878B1,and, for example, include the use of anti-PD-1 antibodies. Exemplaryanti-PD-1 antibodies are described, for example, in U.S. Pat. Nos.8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342,9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and7,488,802. Exemplary anti-PD-1 antibodies include, for example,nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®,Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), andpidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies aredescribed, for example, in U.S. Pat. Nos. 9,273,135, 7,943,743,9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1antibodies include, for example, atezolizumab (Tecentriq®, Genentech),duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (BristolMyers Squibb Co.).

In certain embodiments, a method or composition described herein isadministered in combination with a CTLA-4 inhibitor. In the CTLA-4pathway, the interaction of CTLA-4 on a T-cell with its ligands (e.g.,CD80, also known as B7-1, and CD86) on the surface of an antigenpresenting cells (rather than cancer cells) leads to T-cell inhibition.Exemplary CTLA-4 based immune checkpoint inhibition methods aredescribed in U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227. Exemplaryanti-CTLA-4 antibodies are described in U.S. Pat. Nos. 6,984,720,6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156,7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114,8,784,815, and 8,883,984, International (PCT) Publication Nos.WO98/42752, WO00/37504, and WO01/14424, and European Patent No. EP1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab ortremelimumab.

In certain embodiments, a method or composition described herein isadministered in combination with (i) a PD-1 or PD-L1 inhibitor, e.g., aPD-1 or PD-L1 inhibitor disclosed herein, and (ii) CTLA-4 inhibitor,e.g., a CTLA-4 inhibitor disclosed herein.

In certain embodiments, a method or composition described herein isadministered in combination with an IDO inhibitor. Exemplary IDOinhibitors include 1-methyl-D-tryptophan (known as indoximod),epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205.

Yet other agents that may be used as part of a combination therapy intreating cancer are monoclonal antibody agents that targetnon-checkpoint targets (e.g., herceptin) and non-cytotoxic agents (e.g.,tyrosine-kinase inhibitors).

Yet other categories of anti-cancer agents include, for example: (i) aninhibitor selected from an ALK Inhibitor, an ATR Inhibitor, an A2AAntagonist, a Base Excision Repair Inhibitor, a Bcr-Abl Tyrosine KinaseInhibitor, a Bruton's Tyrosine Kinase Inhibitor, a CDC7 Inhibitor, aCHK1 Inhibitor, a Cyclin-Dependent Kinase Inhibitor, a DNA-PK Inhibitor,an Inhibitor of both DNA-PK and mTOR, a DNMT1 Inhibitor, a DNMT1Inhibitor plus 2-chloro-deoxyadenosine, an HDAC Inhibitor, a HedgehogSignaling Pathway Inhibitor, an IDO Inhibitor, a JAK Inhibitor, a mTORInhibitor, a MEK Inhibitor, a MELK Inhibitor, a MTH1 Inhibitor, a PARPInhibitor, a Phosphoinositide 3-Kinase Inhibitor, an Inhibitor of bothPARP1 and DHODH, a Proteasome Inhibitor, a Topoisomerase-II Inhibitor, aTyrosine Kinase Inhibitor, a VEGFR Inhibitor, and a WEE1 Inhibitor; (ii)an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS;and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

It is understood that the antibody or multi-specific binding proteindisclosed herein, which is designed to activate T lymphocytes, may causeside effects such as neurotoxicity. Accordingly, in certain embodiments,the second therapeutic agent that can be used in combination with theantibody or multi-specific binding protein comprises an agent thatmitigates a side effect of the antibody or multi-specific bindingprotein, e.g., reduces neurotoxicity. In certain embodiments, the secondtherapeutic agent inhibits T cell trafficking, for example, reduces orinhibits immune cells from crossing the blood-brain barrier.Non-limiting examples of such therapeutic agents include antagonists(e.g., antagonistic antibodies) of adhesion molecules on immune cells(e.g., α4 integrin), such as natalizumab. In certain embodiments, thesecond therapeutic agent increases the internalization of asphingosine-1-phosphate (SIP) receptor (e.g., S1PR1 or S1PR5), such asfingolimod or ozanimod. In certain embodiments, the second therapeuticagent is a nitric oxide synthase (NOS) inhibitor, such as ronopterin,cindunistat, A-84643, ONO-1714, L-NOARG, NCX-456, VAS-2381, GW-273629,NXN-462, CKD-712, K_(D)-7040, or guanidinoethyldisulfide. In certainembodiments, the second therapeutic agent is an antagonist of CSF1 orCSF1R, such as pexidartinib, emactuzumab, cabiralizumab, LY-3022855,JNJ-40346527, or MCS110. Additional non-limiting examples of the secondtherapeutic agents include pentosan polysulfate, minocycline,anti-ICAM-1 antibodies, anti-P-selectin antibodies, anti-CD11aantibodies, anti-CD162 antibodies, and anti-IL-6R antibodies (e.g.,tocilizumab).

The amount of the antibody or multi-specific binding protein andadditional therapeutic agent and the relative timing of administrationmay be selected in order to achieve a desired combined therapeuticeffect. For example, when administering a combination therapy to apatient in need of such administration, the therapeutic agents in thecombination, or a pharmaceutical composition or compositions comprisingthe therapeutic agents, may be administered in any order such as, forexample, sequentially, concurrently, together, simultaneously and thelike. Further, for example, an antibody or multi-specific bindingprotein may be administered during a time when the additionaltherapeutic agent(s) exerts its prophylactic or therapeutic effect, orvice versa.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present invention that consist essentially of, or consist of, therecited processing steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components.

Further, it should be understood that elements and/or features of acomposition or a method described herein can be combined in a variety ofways without departing from the spirit and scope of the presentinvention, whether explicit or implicit herein. For example, wherereference is made to a particular compound, that compound can be used invarious embodiments of compositions of the present invention and/or inmethods of the present invention, unless otherwise understood from thecontext. In other words, within this application, embodiments have beendescribed and depicted in a way that enables a clear and conciseapplication to be written and drawn, but it is intended and will beappreciated that embodiments may be variously combined or separatedwithout parting from the present teachings and invention(s). Forexample, it will be appreciated that all features described and depictedherein can be applicable to all aspects of the invention(s) describedand depicted herein.

It should be understood that the expression “at least one of” includesindividually each of the recited objects after the expression and thevarious combinations of two or more of the recited objects unlessotherwise understood from the context and use. The expression “and/or”in connection with three or more recited objects should be understood tohave the same meaning unless otherwise understood from the context.

The use of the term “include,” “includes,” “including,” “have,” “has,”“having,” “contain,” “contains,” or “containing,” including grammaticalequivalents thereof, should be understood generally as open-ended andnon-limiting, for example, not excluding additional unrecited elementsor steps, unless otherwise specifically stated or understood from thecontext.

Where the use of the term “about” is before a quantitative value, thepresent invention also includes the specific quantitative value itself,unless specifically stated otherwise. As used herein, the term “about”refers to a ±10% variation from the nominal value unless otherwiseindicated or inferred.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present invention remainoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

The use of any and all examples, or exemplary language herein, forexample, “such as” or “including,” is intended merely to illustratebetter the present invention and does not pose a limitation on the scopeof the invention unless claimed. No language in the specification shouldbe construed as indicating any non-claimed element as essential to thepractice of the present invention.

The description above describes multiple aspects and embodiments of theinvention. The patent application specifically contemplates allcombinations and permutations of the aspects and embodiments.

EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and is not intended to limit the invention.

Example 1. Characterization of New Anti-Serum Albumin Antibodies

This example describes new anti-serum albumin antibodies CNG-HSA-101 to-120. The amino acid sequences of these antibodies are provided in Table1 above.

CNG-HSA-101 to -120 were optimized from parental antibodies CNG-HSA-1, asingle domain antibody, by introducing diversities into the heavy chainvariable region, generating random mutations by error prone PCR, andshuffling the VH fragments. The antibody clones were selected forimproved binding affinity to biotinylated human serum albumin relativeto the parental antibody. Additionally, a thermal selection pressure wasemployed for the VH shuffle optimization cycle. Thermal selectionpressures were applied by incubating the libraries at varioustemperatures and then selecting for antibodies that retained antigenbinding following thermal incubation. The selected antibodies were thenproduced from yeast cells and purified using a protein A column.

The binding affinity of the antibodies to isolated serum albumin wasmeasured by surface plasmon resonance using a ForteBio Octet HTX systemas previously described (see, e.g., Estep et al, High throughputsolution-based measurement of antibody-antigen affinity and epitopebinning Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinitymeasurements were performed by loading the heavy chain antibodies(HCAbs) on-line onto AHC sensors. The sensors were equilibrated off-linein assay buffer for 30 minutes and then monitored on-line for 60 secondsfor baseline establishment. The sensors with loaded HCAbs were exposedto 100 nM human serum albumin for 3 minutes, and subsequentlytransferred to assay buffer for 3 minutes for off-rate measurement. Allkinetics were analyzed using the 1:1 binding model.

The melting temperature (Tm) of the VHH fragments were measured bydynamic scanning fluorimetry (DSF). Briefly, 10 μL of 20× Sypro Orangedye is added to 20 μL of 0.2-1 mg/mL HCAb. A BioRad CFX96 RT PCR machineis used to raise the sample plate temperature from 40° to 95° C. at 0.5°C. increments, with 2 minutes equilibration at each temperature. Thenegative of the first derivative for the raw data is used to extract Tm.

TABLE 5 Binding of Anti-Serum Albumin Antibodies to Serum Albumin andProtein A Affinity to Ratio Human Affinity to Affinity to of SerumCynomolgus Mouse Serum MSA Albumin Serum Albumin -K_(D) Affinity to(HSA) Albumin (MSA) to Protein A Melting K_(D) k_(off) K_(D) k_(off)K_(D) k_(off) HSA- K_(d) koff Temperature Construct (M) (1/s) (M) (1/s)(M) (1/s) K_(D) (M) (1/s) (° C.) CNG- 1.19E−08 1.77E−03 9.65E−091.39E−03 1.72E−07 2.16E−02 14.47 2.56E−09 4.74E−04 55.5 HSA-1 CNG-5.07E−09 7.16E−04 3.58E−09 5.32E−04 1.61E−08 3.41E−03 3.17 1.84E−092.00E−04 65.5 HSA-101 CNG- 4.89E−09 7.48E−04 3.65E−09 5.68E−04 4.14E−087.38E−03 8.46 1.70E−09 2.00E−04 66.5 HSA-102 CNG- 4.89E−09 7.42E−043.62E−09 5.41E−04 1.51E−08 3.24E−03 3.09 2.02E−09 2.21E−04 66.0 HSA-103CNG- 7.71E−09 1.12E−03 6.10E−09 8.74E−04 5.06E−08 1.05E−02 6.57 1.70E−092.00E−04 63.5 HSA-104 CNG- 1.52E−08 2.00E−03 1.27E−08 1.63E−03 1.54E−072.06E−02 10.12 2.76E−09 2.97E−04 64.0 HSA-105 CNG- 1.70E−08 2.97E−031.44E−08 2.38E−03 5.78E−08 1.24E−02 3.41 N.B.¹ 65.0 HSA-106 CNG-1.68E−08 2.38E−03 1.33E−08 1.82E−03 4.56E−08 8.51E−03 2.72 3.09E−093.33E−04 59.0 HSA-107 CNG- 6.19E−09 9.58E−04 5.27E−09 7.94E−04 8.06E−091.85E−03 1.30 2.94E−09 2.94E−04 64.0 HSA-108 CNG- 3.50E−09 5.66E−043.10E−09 5.03E−04 1.19E−08 2.84E−03 3.40 1.78E−09 2.00E−04 63.5 HSA-109CNG- 5.70E−09 8.93E−04 5.20E−09 7.59E−04 3.89E−08 8.38E−03 6.82 3.67E−093.78E−04 57.5 HSA-110 CNG- 8.35E−09 1.17E−03 6.13E−09 8.84E−04 1.02E−082.31E−03 1.23 2.84E−09 2.00E−04 59.0 HSA-111 CNG- 8.45E−09 1.36E−037.43E−09 1.15E−03 6.67E−08 1.30E−02 7.90 3.44E−09 3.75E−04 54.5 HSA-112CNG- 1.54E−08 2.14E−03 1.21E−08 1.70E−03 5.20E−08 1.01E−02 3.38 1.70E−092.00E−04 62.5 HSA-113 CNG- 1.47E−08 2.21E−03 1.20E−08 1.73E−03 1.75E−083.80E−03 1.20 N.D.² 51.0 HSA-114 CNG- 2.96E−09 4.41E−04 3.37E−096.18E−04 6.43E−09 1.32E−03 2.17 N.D. 56.0 HSA-115 CNG- 4.65E−09 6.99E−043.85E−09 5.97E−04 4.56E−09 1.08E−03 0.98 1.75E−09 2.00E−04 64.0 HSA-116CNG- 5.56E−09 7.93E−04 4.05E−09 6.08E−04 4.48E−08 8.77E−03 8.07 1.78E−092.00E−04 60.5 HSA-117 CNG- 9.36E−09 1.30E−03 6.65E−09 1.02E−03 1.20E−082.74E−03 1.28 N.D. 58.0 HSA-118 CNG- 7.36E−09 1.09E−03 5.73E−09 8.64E−044.11E−08 8.54E−03 5.59 N.D. 53.5 HSA-119 CNG- 3.15E−09 5.07E−04 2.60E−094.22E−04 4.32E−09 1.07E−03 1.37 N.D. 66.0 HSA-120 ¹N.B. means no bindingwas detected under the conditions of this assay. ²N.D. means notdetermined.

As shown in Table 5, CNG-HSA-101 to -120 showed higher binding affinityto human serum albumin, cynomolgus serum albumin, mouse serum albumin,and/or protein A than CNG-HSA-1. In particular, all these antibodiesbound mouse serum albumin with lower K_(D) values than CNG-HSA-1.CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108,CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115,CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, and CNG-HSA-120bound human serum albumin and cynomolgus serum albumin with lower K_(D)values than CNG-HSA-1. CNG-HSA-101, CNG-HSA-103, CNG-HSA-106,CNG-HSA-107, CNG-HSA-108, CNG-HSA-109, CNG-HSA-111, CNG-HSA-113,CNG-HSA-114, CNG-HSA-115, CNG-HSA-116, CNG-HSA-118, and CNG-HSA-120bound mouse serum albumin with a K_(D) value less than 4-fold higherthan the K_(D) with which the same antibody bound human serum albumin.CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113,CNG-HSA-116, and CNG-HSA-117 bound protein A with lower K_(D) valuesthan CNG-HSA-1. CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104,CNG-HSA-105, CNG-HSA-106, CNG-HSA-108, CNG-HSA-109, CNG-HSA-113,CNG-HSA-116, CNG-HSA-117, and CNG-HSA-120 showed a melting temperaturegreater than or equal to 60° C., among which CNG-HSA-101, CNG-HSA-102,CNG-HSA-103, CNG-HSA-106, and CNG-HSA-120 showed a melting temperaturegreater than or equal to 65° C.

Example 2. Production of Multi-Specific Binding Proteins

This example describes the production and purification of multi-specificbinding proteins.

Nucleic acids encoding single-chain multi-specific binding proteins (seeTable 6) were constructed and codon optimized for expression in humancells and cloned into a mammalian expression vector following standardprocedures. Following sequence verification, the expression vectors, inthe form of plasmids, were prepared in sufficient quantity fortransfection using Plasmid Plus purification kits (Qiagen). Humanembryonic kidney 293 (HEK 293) cells were passaged to appropriatedensity for transient transfection. Cells were transiently transfectedwith the expression vectors and cultured for six days.

The amino acid sequences of the various multi-specific binding proteinsare summarized in Table 6. Constructs tAb0027 to tAb0032 each containedan anti-CD19 scFv having the amino acid sequence set forth in SEQ ID NO:9, an anti-CD3 scFv having the amino acid sequence set forth in SEQ IDNO: 105, and an anti-HSA sdAb having the amino acid sequence set forthin SEQ ID NO: 121. Constructs tAb0033 to tAb0038 each contained ananti-CD19 scFv having the amino acid sequence set forth in SEQ ID NO:18, an anti-CD3 scFv having the amino acid sequence set forth in SEQ IDNO: 105, and an anti-HSA sdAb having the amino acid sequence set forthin SEQ ID NO: 121.

TABLE 6 Exemplary Multi-specific Binding Proteins Construct FormatAmino Acid Sequence tAb0027 CD19:CD3:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQ HSAGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIKGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSHHHHHHHHHH (SEQ ID NO: 303) tAb0029 CD3:CD19:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQK HSAPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSGGGGSGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIKGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSHHHHHHHHHH (SEQ ID NO: 304) tAb0030 CD3:HSA:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQK CD19PGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIKHHHHHHHHHH (SEQ ID NO: 305) tAb0031 HSA:CD3:EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL CD19EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSGGGGSGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIKHHHHHHHHHH (SEQ ID NO: 306) tAb0032 HSA:CD19EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL :CD3EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIKGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSHHHHHHHHHH (SEQ ID NO: 307) tAb0033 CD19:CD3:QVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGK HSAGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSVYPFTFGQGTKLEIKRGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSHHH HHHHHHH (SEQ ID NO: 308)tAb0034 CD19:HSA: QVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGK CD3GLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSVYPFTFGQGTKLEIKRGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSHHH HHHHHHH (SEQ ID NO: 309)tAb0035 CD3:CD19: DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQK HSAPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSGGGGSGGGSQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSVYPFTFGQGTKLEIKRGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSHHH HHHHHHH (SEQ ID NO: 310)tAb0036 CD3:HSA: DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQK CD19PGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSVYPFTFGQGTKLEIKRHHH HHHHHHH (SEQ ID NO: 311)tAb0037 HSA:CD3: EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL CD19EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSGGGGSGGGSQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSVYPFTFGQGTKLEIKRHHH HHHHHHH (SEQ ID NO: 312)tAb0038 HSA:CD19: EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL CD3EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSVYPFTFGQGTKLEIKRGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSSHHH HHHHHHH (SEQ ID NO: 313)tAb0042 CD3:HSA: EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKG CD19LEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGER

The cultures were harvested by centrifugation at 4000 rpm, and thesupernatant filtered through a 0.22 mm filter. The multi-specificbinding proteins, which carried a 10×His tag at the C-terminus, werepurified in two steps. The first step was Nickel affinity chromatographywith elution using PBS containing 400 mM imidazole. The second step wassize exclusion chromatography with elution in PBS (phosphate bufferedsaline) pH7.2. Multi-specific binding protein concentrations weredetermined by UV spectroscopy, and the protein samples were concentratedwhen necessary. The purity of the proteins was determined by sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and highperformance liquid chromatography (HPLC). Specifically, HPLC wasperformed on an Agilent 1100 series instrument using MabPac sizeexclusion column run in PBS at 0.2 mL/min. The fractions with an elutiontime of about 225-240 minutes were collected for furthercharacterization.

As noted above, the constructs produced contained an anti-CD19 scFvhaving the amino acid sequence set forth in SEQ ID NO: 9 or 18. Thebinding affinity of the two CD19 binding domains to CD19 were measuredby SPR using a monomeric CD19 extracellular domain and a dimeric CD19extracellular domain fused with human IgG1 Fc. Binding kineticparameters were measured using a ForteBio instrument generally aspreviously described (see, Estep et al. (2013) MAbs, 5(2): 270-78). Whenmeasured with the monomeric CD19 protein, the K_(D) value of the CD19binding domain having the sequence of SEQ ID NO: 9 was 7 nM, and theK_(D) value of the CD19 binding domain having the sequence of SEQ ID NO:18 was 11 nM. When measured with the dimeric CD19 protein, the K_(D)value of the CD19 binding domain having the sequence of SEQ ID NO: 9 was5 nM, and the K_(D) value of the CD19 binding domain having the sequenceof SEQ ID NO: 18 was 15 nM.

Example 3. Multi-Specific Binding Proteins Induce T Cell CytotoxicityAgainst CD19⁺ Target Cells

This example describes the cytotoxic activity of multi-specific bindingproteins.

The T cell redirection activity of multi-specific binding proteins andBiTE proteins were evaluated using the KILR Raji Cell Model. Briefly,pan T cells were isolated from primary human PBMCs from a single healthydonor by negative selection using a commercial kit (e.g. Easy Sep HumanT Cell Enrichment Kit, StemCell Technologies). T cells were maintainedin RPMI 1640 medium supplemented with 10% serum and 300 IU/mL IL-2 toexpand T cells. The harvested T cells were washed twice to remove anyserum.

KILR Raji cells, which expressed CD19 on the surface, were used astarget cells. To opsonize the target cells, each multi-specific bindingprotein or BiTE (see Table 6) was incubated with the target cells for 30minutes at 37° C. in RPMI 1640 medium supplemented with 5% heatinactivated low IgG fetal bovine serum andpenicillin-streptomycin-glutamine. The proteins were added in serialdilution at 10 different doses, with each dose run in duplicate. Humanserum albumin was added to the medium of certain samples at a finalconcentration of 15 mg/mL. Selective proteins were also evaluated withKILR SKOV3 cells, which were CD19-negative, as negative controls.

After opsonization, the target cells were incubated with the pan T cellsat an effector-to-target (E:T) ratio of 10:1 for 6 hours at 37° C.Killing of the KILR Raji cells resulted in release of a labeledhousekeeping protein from these cells into the medium, which wasquantified by addition of a KILR detection reagent (DiscoverX). Theluminescence signals from all wells were read on an Envision platereader. Spontaneous release and total lysis controls were included oneach plate to allow calculation of percent killing.

Percent killing was calculated from the luminescence signal values usingthe following formula:

% killing=(value from test protein sample−mean value from spontaneousrelease control)/(mean value from total lysis control−mean value fromspontaneous release control)×100.

The EC50 values were calculated from the percent killing by fitting witha dose-response curve using the GraphPad Prism software.

Table 7 lists the EC50 values of T cell-redirected killing in theabsence and presence of human serum albumin for exemplary multi-specificbinding proteins and comparator anti-CD19 BiTE protein. No substantialkilling was observed with the CD19-negative KILR SKOV3 cells.

TABLE 7 Cytotoxic Activity of Multi-specific Binding Proteins EC50(pg/mL) Fold Construct Format −HSA +HSA change tAb0027 CD19:CD3:HSA 2.3367.9 29.1 tAb0029 CD3:CD19:HSA 5.48 188.1 34.3 tAb0030 CD3:HSA:CD19 3.17141.7 44.7 tAb0031 HSA:CD3:CD19 6.55 189.3 28.9 tAb0032 HSA:CD19:CD35.71 88.4 15.5 tAb0033 CD19:CD3:HSA 20.4 1294 63.4 tAb0034 CD19:HSA:CD359.3 2376 40.1 tAb0035 CD3:CD19:HSA 51.4 2184 42.5 tAb0036 CD3:HSA:CD19103.8 4195 40.4 tAb0037 HSA:CD3:CD19 175.1 2151 12.3 tAb0038HSA:CD19:CD3 52.6 288 5.5 tAb0042 CD3:HSA:CD19 69.9 13290 190.1blinatumomab CD19:CD3 2854 10750 3.8

As shown in Table 7, the multi-specific binding proteins containing theanti-CD19 scFv having the amino acid sequence of SEQ ID NO: 9 showedstronger cytotoxic activity than those containing the anti-CD19 scFvhaving the amino acid sequence of SEQ ID NO: 18, regardless of theconstruct format, CD3 binding domain, HSA binding domain, and thepresence or absence of HSA in the assay medium. From this data, it iscontemplated that constructs containing this anti-CD19 scFv with thehigher binding affinity to CD19 will demonstrate stronger therapeuticactivity than constructs containing the other anti-CD19 scFv with thelower binding affinity.

Furthermore, all the multi-specific binding proteins tested showed lowerEC₅₀ value (namely, stronger ability to induce cytotoxicity) in theabsence of HSA than in the presence of HSA. Without wishing to be boundby theory, it appears that the presence of HSA causes a change in theprotein complex, which was specific to the multi-specific bindingproteins containing an HSA binding domain, rather than a nonspecificeffect as observed with blinatumomab. The ratio of the EC₅₀ value in thepresence of HSA to the EC₅₀ value in the absence of HSA, also called“fold change” herein, was used to assess the effect of HSA on thepotential therapeutic activity of the multi-specific binding protein. Asshown in Table 7, the construct formats with the HSA binding domainN-terminal to both the CD19 binding domain and the CD3 binding domain(namely, tAb0031, tAb0032, tAb0037, and tAb0038) showed lower foldchanges than the other construct formats, regardless of which CD19binding domain was used in the construct.

Furthermore, among the constructs having the same CD19 binding domain,CD3 binding domain, and HSA binding domain, the constructs in theCD19:CD3:HSA format (i.e., the CD19 binding domain positioned N-terminalto the CD3 binding domain, and the CD3 binding domain positionedN-terminal to the HSA binding domain), namely, tAb0027 and tAb0033,showed the lowest or second lowest EC₅₀ values both in the absence andin the presence of HSA.

Example 4. Cytotoxicity of Multi-Specific Binding Proteins Against CD19⁺Target Cells

This example provides alternative methods for determining the cytotoxicactivity of a multi-specific binding protein.

The multi-specific binding proteins disclosed herein can be evaluated inin vitro assays on their mediation of T cell dependent cytotoxicity to Bcell antigen positive target cells. For example, the CD19-bindingmulti-specific binding protein disclosed herein is evaluated in in vitroassays on its mediation of T cell dependent cytotoxicity to CD19⁺ targetcells.

Fluorescence labeled CD19⁺ MEC-1 cells (a CD19+ human chronic B cellleukemia cell line) are incubated with isolated PBMC of random donors orCB15 T-cells (standardized T-cell line) as effector cells in thepresence of the CD19-binding multi-specific binding protein. Afterincubation for 4 hours at 37° C. in a humidified incubator, the releaseof the fluorescent dye from the target cells into the supernatant isdetermined in a spectrofluorimeter. Target cells incubated without theCD19-binding multi-specific binding protein and target cells totallylysed by the addition of saponin at the end of the incubation serve asnegative and positive controls, respectively. Based on the measuredremaining living target cells, the percentage of specific cell lysis canbe calculated according to the following formula: [1−(number of livingtargets_((sample))/number of living targets_((spontaneous))]×100%.Sigmoidal dose response curves and EC₅₀ values are calculated bynon-linear regression/4-parameter logistic fit using the GraphPadSoftware. The lysis values obtained for a given multi-specific bindingprotein concentration are used to calculate sigmoidal dose-responsecurves by 4 parameter logistic fit analysis using the Prism software. Itis expected that the target cell lysis rate induced by CD19-bindingmulti-specific binding protein is higher than the target cell lysis rateinduced by similar constructs lacking either a CD19-binding domain or aCD3-binding domain.

Alternatively, a human T-cell dependent cellular cytotoxicity (TDCC)assay is used to measure the ability of the multi-specific bindingprotein to direct T cells to kill tumor cells (Nazarian et al. 2015, JBiomol. Screen, 20:519-27). In this assay, T cells and target cancercell line cells are mixed together at a 10:1 ratio in a 384 wells plate,and varying amounts of the multi-specific binding proteins are added.After 48 hours, the T cells are washed away leaving attached to theplate target cells that were not killed by the T cells. To quantitatethe remaining viable cells, CellTiter-Glo® Luminescent Cell ViabilityAssay (Promega) is used. It is contemplated that the killing rate ofB-cell antigen expressing cancer cell induced by CD19-bindingmulti-specific binding protein will be higher than that induced bysimilar constructs lacking either a CD19-binding domain or a CD3-bindingdomain and/or other negative control molecules.

Example 5. Pharmacokinetics of Multi-Specific Binding Proteins with HSABinding Domain

This example is designed to determine the pharmacokinetics ofmulti-specific binding proteins.

Multi-specific binding proteins containing a domain that binds CD19, adomain that binds CD3, and a domain that binds serum albumin are testedin the cynomolgus monkey in the context of pharmacokinetic (PK) studiesto evaluate the serum elimination time of the multi-specific bindingprotein.

The multi-specific binding proteins are administered as intravenousbolus or intravenous infusion. The multi-specific binding proteins areadministered in a dose-linear, pharmacokinetic relevant range of 0.5μg/kg to 3 μg/kg, 6 μg/kg, 12 μg/kg, and 15 μg/kg, respectively. Forpurposes of comparability, the serum concentrations of themulti-specific binding proteins are does-normalized and molecularweight-normalized (described in nmol).

For each multi-specific binding protein, a group of at least two tothree animals are used. Blood samples are collected and serum isprepared for determination of serum concentrations of the multi-specificbinding proteins. Serum multi-specific binding protein levels aremeasured using an immunoassay. The assay is performed by capturing themulti-specific binding protein via its CD19-binding domain, while anantibody directed against the CD3-binding domain of the multi-specificbinding protein is used for detection. The serum concentration-timeprofiles are used to determine PK parameters using known analyticalmethods such as those described in Ritschel W A and Kearns G L, 1999,IN: Handbook Of Basic Pharmacokinetics Including Clinical Applications,5th edition, American Pharmaceutical Assoc., Washington, D.C. andsoftwares such as WinNonlin software (WinNonlin® Professional V. 3.1WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View,Calif.).

Alternatively, the serum half-life of the various multi-specific bindingproteins containing the serum albumin binding domain is compared to thatof control constructs capable of binding CD19 and CD3 but lacking aserum albumin binding domain by including in the experiment anothercynomolgus monkey group that receives the control constructs. Additionaldomains can be included such that the control constructs are similar insize to the multi-specific binding proteins.

It is expected that CD19-binding multi-specific binding protein willhave significantly longer serum half-life compared to similar constructscapable of binding CD19 and CD3 but lacking a serum albumin bindingdomain and/or other negative control molecules.

Example 6. Determination of Antigen Affinity by Flow Cytometry

This example is designed to determine the affinity of a multi-specificbinding protein to an antigen.

Various multi-specific binding proteins disclosed herein are tested fortheir binding affinities to human CD3⁺ cells and the corresponding Bcell surface antigen positive cells, such as human CD19⁺ cells. Themulti-specific binding proteins are also tested for their bindingaffinities to cynomolgus CD3⁺ cells and the corresponding B cell surfaceantigen positive cells, such as cynomolgus CD19⁺ cells.

CD3⁺ and CD19⁺ cells are incubated with 100 μL of serial dilutions ofthe multi-specific binding protein. After washing three times with FACSbuffer the cells are incubated with 0.1 mL of 10 pg/mL mouse monoclonalanti-idiotype antibody in the same buffer for 45 mins on ice. After asecond washing cycle, the cells are incubated with 0.1 mL of 15 pg/mLFITC-conjugated goat anti-mouse IgG antibodies under the same conditionsas before. As a control, cells are incubated with the anti-His IgGfollowed by the FITC-conjugated goat anti-mouse IgG antibodies withoutthe multi-specific binding protein. The cells are then washed again andresuspended in 0.2 mL of FACS buffer containing 2 pg/mL propidium iodide(PI) in order to exclude dead cells. The fluorescence of 1×10⁴ livingcells is measured using a commercially available flow cytometer andsoftware. Mean fluorescence intensities of the cell samples arecalculated using software such as CXP software (Beckman-Coulter,Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach,Germany). K_(D) values for one-site binding can be calculated usingnormalized fluorescence intensity values with known computationalequations such as those supplied in the GraphPad Prism software(GraphPad Software, La Jolla Calif. USA). CD3 binding affinity andcross-reactivity are evaluated in titration and flow cytometricexperiments on CD3⁺ Jurkat cells and the cynomolgus CD3⁺ HSC-F cellline. CD19 binding and cross-reactivity are assessed on the human CD19⁺tumor cell lines. The K_(D) ratio of cross-reactivity can be calculatedusing the K_(D) values determined on the CHO cell lines expressingeither recombinant human or recombinant cynomolgus antigens.

Example 7. Cytokine Production Induced by Multi-Specific BindingProteins

This example is designed to determine the ability of a multi-specificbinding protein to induce cytokine production from immune cells.

AlphaLISA assays (Perkin Elmer) for TNFα and Interferon γ are used toobtain evidence that T cells are activated by the multi-specific bindingproteins of current invention, such as CD19-binding multi-specificbinding protein, in the presence of target cells, such as CD19⁺ B cells.For this assay, primary human T cells and human tumor cells expressing Bcell surface antigen are incubated in the presence of the CD19-bindingmulti-specific binding protein as described under cytotoxicity assays.After 48 hours of incubation, 2 microliter aliquots of the assaysupernatants are analyzed according to the manufacturer's instructions.It is contemplated that the TNFα or Interferon γ level induced byCD19-binding multi-specific binding protein is higher than that inducedby similar constructs lacking either a CD19-binding domain or aCD3-binding domain and/or other negative control molecules.

INCORPORATION BY REFERENCE

All publications and patents cited throughout the text of thisspecification (including all patents, patent applications, scientificpublications, manufacturer's specifications, instructions, etc.),whether supra or infra, are hereby incorporated by reference in theirentirety for all purposes. To the extent the material incorporated byreference contradicts or is inconsistent with this specification, thespecification will supersede any such material.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. An antigen-binding site that binds human serumalbumin, comprising a VH comprising complementarity determining regionsHCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprisethe amino acid sequences of SEQ ID NOs: 184, 409, and 411, respectively,but not SEQ ID NOs: 129, 133, and 135, respectively.
 2. Theantigen-binding site of claim 1, wherein the HCDR1, HCDR2, and HCDR3comprise the amino acid sequences of SEQ ID NOs: 184, 185, and 187,respectively, but not SEQ ID NOs: 129, 133, and 135, respectively. 3.The antigen-binding site of claim 1, wherein the HCDR1, HCDR2, and HCDR3comprise the amino acid sequences of SEQ ID NOs: 189, 190, and 192,respectively, but not SEQ ID NOs: 129, 133, and 135, respectively. 4.The antigen-binding site of any one of claims 1-3, wherein the HCDR1,HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 189,193, and 195, respectively, but not SEQ ID NOs: 129, 133, and 135,respectively.
 5. The antigen-binding site of claim 4, wherein the HCDR1,HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 123,124, and 126, respectively.
 6. The antigen-binding site of any one ofclaims 1-5, wherein the VH comprises an amino acid sequence at least85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:121.
 7. The antigen-binding site of any one of claims 1-6, wherein theVH comprises the amino acid sequence of SEQ ID NO:
 121. 8. Theantigen-binding site of any one of claims 1-7, wherein theantigen-binding site binds human serum albumin with a K_(D) lower thanor equal to 10 nM.
 9. The antigen-binding site of any one of claims 1-8,wherein the antigen-binding site binds protein A with a K_(D) lower thanor equal to 2 nM.
 10. The antigen-binding site of any one of claims 1-9,wherein the antigen-binding site has a melting temperature greater thanor equal to 60° C.
 11. A multi-specific binding protein comprising: (a)a first antigen-binding site that binds a first target protein expressedon a target cell; (b) a second antigen-binding site that binds a secondtarget protein expressed on an immune effector cell; and (c) a thirdantigen-binding site that binds human serum albumin, wherein the thirdantigen-binding site is an antigen-binding site of any one of claims1-10.
 12. The multi-specific binding protein of claim 11, wherein thefirst antigen-binding site binds human CD19.
 13. The multi-specificbinding protein of claim 11 or 12, wherein the second antigen-bindingsite binds human CD3.
 14. The multi-specific binding protein of any oneof claims 11-13, wherein the multi-specific binding protein comprises asingle polypeptide chain.
 15. The multi-specific binding protein ofclaim 14, wherein the third antigen-binding site is not positionedbetween the first antigen-binding site and the second antigen-bindingsite in the polypeptide chain.
 16. The multi-specific binding protein ofclaim 15, wherein the third antigen-binding site is positionedN-terminal to both the first antigen-binding site and the secondantigen-binding site in the polypeptide chain.
 17. The multi-specificbinding protein of claim 16, wherein the third antigen-binding site ispositioned N-terminal to the first antigen-binding site, and the firstantigen-binding site is positioned N-terminal to the secondantigen-binding site in the polypeptide chain.
 18. The multi-specificbinding protein of claim 16, wherein the third antigen-binding site ispositioned N-terminal to the second antigen-binding site, and the secondantigen-binding site is positioned N-terminal to the firstantigen-binding site in the polypeptide chain.
 19. The multi-specificbinding protein of claim 15, wherein the third antigen-binding site ispositioned C-terminal to both the first antigen-binding site and thesecond antigen-binding site in the polypeptide chain.
 20. Themulti-specific binding protein of claim 19, wherein the firstantigen-binding site is positioned N-terminal to the secondantigen-binding site, and the second antigen-binding site is positionedN-terminal to the third antigen-binding site in the polypeptide chain.21. The multi-specific binding protein of claim 19, wherein the secondantigen-binding site is positioned N-terminal to the firstantigen-binding site, and the first antigen-binding site is positionedN-terminal of the third antigen-binding site in the polypeptide chain.22. The multi-specific binding protein of claim 14, wherein the firstantigen-binding site is positioned N-terminal to the thirdantigen-binding site, and the third antigen-binding site is positionedN-terminal to the second antigen-binding site in the polypeptide chain.23. The multi-specific binding protein of claim 14, wherein the secondantigen-binding site is positioned N-terminal to the thirdantigen-binding site, and the third antigen-binding site is positionedN-terminal binding protein the first antigen-binding site in thepolypeptide chain.
 24. The multi-specific binding protein of any one ofclaims 11-23, wherein the first antigen-binding site comprises asingle-chain variable fragment (scFv).
 25. The multi-specific bindingprotein of any one of claims 11-24, wherein the third antigen-bindingsite comprises a single domain antibody (sdAb).
 26. The multi-specificbinding protein of any one of claims 11-25, wherein the secondantigen-binding site comprises an scFv.
 27. The multi-specific bindingprotein of any one of claims 13-26, wherein the second antigen-bindingsite binds human CD3ε.
 28. The multi-specific binding protein of claim27, wherein the second antigen-binding site binds human CD3ε with aK_(D) in the range of 1-100 nM.
 29. The multi-specific binding proteinof any one of claims 13-28, wherein the second antigen-binding sitecomprises a VH comprising complementarity determining regions HCDR1,HCDR2, and HCDR3, and a VL comprising complementarity determiningregions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ IDNOs: 415, 416, 418, 419, 420, and 421, respectively.
 30. Themulti-specific binding protein of claim 29, wherein the VH comprises anamino acid sequence at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 412, and the VL comprises an aminoacid sequence at least 85%, at least 90%, at least 95%, at least 99%, or100% identical to SEQ ID NO:
 413. 31. The multi-specific binding proteinof claim 29 or 30, wherein the antigen-binding site comprises the aminoacid sequence of SEQ ID NO: 422 or
 423. 32. The multi-specific bindingprotein of any one of claims 13-28, wherein the second antigen-bindingsite comprises a VH comprising complementarity determining regionsHCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determiningregions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ IDNOs: 415, 416, 426, 419, 420, and 421, respectively.
 33. Themulti-specific binding protein of claim 32, wherein the VH comprises anamino acid sequence at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 424, and the VL comprises an aminoacid sequence at least 85%, at least 90%, at least 95%, at least 99%, or100% identical to SEQ ID NO:
 413. 34. The multi-specific binding proteinof claim 32 or 33, wherein the antigen-binding site comprises the aminoacid sequence of SEQ ID NO: 427 or
 428. 35. The multi-specific bindingprotein of any one of claims 13-28, wherein the second antigen-bindingsite comprises a VH comprising complementarity determining regionsHCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determiningregions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ IDNOs: 415, 431, 418, 419, 420, and 432, respectively.
 36. Themulti-specific binding protein of claim 35, wherein the VH comprises anamino acid sequence at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 429, and the VL comprises an aminoacid sequence at least 85%, at least 90%, at least 95%, at least 99%, or100% identical to SEQ ID NO:
 430. 37. The multi-specific binding proteinof claim 35 or 36, wherein the antigen-binding site comprises the aminoacid sequence of SEQ ID NO: 433 or
 434. 38. The multi-specific bindingprotein of any one of claims 11-37, wherein at least two adjacentantigen-binding sites are connected by a peptide linker.
 39. Themulti-specific binding protein of claim 38, wherein each of the adjacentantigen-binding sites are connected by a peptide linker.
 40. Themulti-specific binding protein of claim 38 or 39, wherein the peptidelinker comprises the amino acid sequence of SEQ ID NO: 298, 299, or 302.41. The multi-specific binding protein of claim 38 or 39, wherein thepeptide linker consists of the amino acid sequence of SEQ ID NO: 298,299, or
 302. 42. The multi-specific binding protein of any one of claims11-41, wherein the multi-specific binding protein does not comprise anantibody Fc region.
 43. The multi-specific binding protein of any one ofclaims 11-42, wherein the molecular weight of the multi-specific bindingprotein is at least 65 kD.
 44. The multi-specific binding protein of anyone of the preceding claims, wherein the serum half-life of themulti-specific binding protein is at least 24, 36, 48, or 60 hours. 45.An antibody comprising the antigen-binding site of any one of claims1-10.
 46. A pharmaceutical composition comprising: (a) themulti-specific binding protein of any one of claims 11-44 or theantibody of claim 45; and (b) a pharmaceutically acceptable carrier. 47.An isolated polynucleotide encoding the multi-specific binding proteinof any one of claims 11-44 or the antibody of claim
 45. 48. A vectorcomprising the polynucleotide of claim
 47. 49. A recombinant host cellcomprising the polynucleotide of claim 47 or the vector of claim
 48. 50.A method of producing a multi-specific binding protein or an antibody,the method comprising culturing the host cell of claim 49 under suitableconditions that allow expression of the multi-specific binding proteinor the antibody.
 51. The method of claim 50, further comprisingisolating the multi-specific binding protein or the antibody.
 52. Themethod of claim 51, further comprising formulating the isolatedmulti-specific binding protein or antibody with a pharmaceuticallyacceptable carrier.
 53. A method of stimulating an immune responseagainst a target cell, the method comprising exposing the cell and a Tlymphocyte to the multi-specific binding protein of any one of claims11-44, the antibody of claim 45, or the pharmaceutical composition ofclaim
 46. 54. A method of treating a hematologic cancer in a subject inneed thereof, the method comprising administering to the subject aneffective amount of the multi-specific binding protein of any one ofclaims 11-44, the antibody of claim 45, or the pharmaceuticalcomposition of claim
 46. 55. The method of claim 54, wherein thehematologic cancer is a B-cell hematologic malignancy.
 56. A complexcomprising a T cell expressing CD3, a B cell expressing CD19, and themulti-specific binding protein of any one of claims 13-44, wherein themulti-specific binding protein simultaneously bind both the T cell andthe B cell.
 57. The complex of claim 56, further comprising serumalbumin.